Monoclonal antibodies and their bispecific variants have demonstrated efficacy in diagnostic and therapeutic applications in various fields over the past decades, including autoimmune diseases, cancer, and infectious diseases. However, the targeting of tumor-associated antigens by high-affinity antibodies is typically confined to surface-exposed or secreted proteins, despite the fact that the majority of disease-related altered protein-protein interactions occur in the cytosol. Antibodies that are capable of overcoming the cell membrane barrier and accumulating in the cytosol are classified as cytosol-penetrating antibodies. In the majority, cell binding occurs through electrostatic interactions with the extracellular matrix, leading to a typically non-specific process of cytosol penetration. The present work is focused on the establishment of detection methods for the verification of cytosolic transport of proteins and peptides, as well as the generation and characterization of conditionally activatable, tumor cell-specific, cell-penetrating peptides and antibodies. The initial study in this cumulative work aimed to characterize a conditionally activatable, cytosol-penetrating antibody, in the following referred to as CPAb, and to compare it with the well-known cytosol-penetrating antibody cytotransmab. First, the function and relevance of the HSPG binding motif for the binding of the unmasked CPAb on target cells and the subsequent internalization were verified. The cytosol-penetrating capabilities of the CPAbs were demonstrated by two orthogonal detection methods resulting in cytosol penetration rates that were either similar to or higher than those observed with cytotransmab. The detection methods were based on a fragmented luciferase and a truncated variant of the catalytic domain of Pseudomonas exotoxin A. An immune library derived from an immunized chicken was employed, utilizing yeast surface display (YSD) in conjunction with fluorescence-activated cell sorting (FACS), to facilitate the sorting and isolation of affinity binders directed against the binding domain of scFv-formatted CPAb, thereby enabling the generation of affinity-based masking units. The generation of two masked and conditionally activatable CPAb variants was achieved by the N-terminal attachment of the masking units to the light chain of the antibody via a cleavable linker sequence. This linker sequence was designed to be cleaved in the tumor microenvironment (TME) by the insertion of a recognition sequence of the in TME overexpressed matrix metalloprotease-9 (MMP-9). The inhibitory function of the mask on binding and internalization, as well as its influence on cytosol-penetrating properties, were investigated and confirmed through fluorescence labeling and cytosol penetration assays in vitro. The cleavage of the mask resulted in the complete regeneration of the cytosol-penetrating properties. The masked CPAb variant represents the first masked, cytosol-penetrating antibody whose capacity for cytosolic translocation, in the function of a transport protein for cargo proteins, can be simultaneously conditionally activated in the TME. The second investigation was conducted with the objective of generation and characterization of tumor cell-specific and cell-penetrating antibodies, based on the previously generated masked CPAb variant. Three distinct bispecific antibodies were engineered through the utilization of the knobs-into-holes methodology, with the masked CPAb positioned on one arm and a tumor-associated antigen (TAA) binding antibody fragment on the second arm. To facilitate the release of the antibody from the receptor following clathrin-mediated endocytosis, an additional furin cleavage site was inserted between the TAA-binding antibody fragment (anti-HER2, anti-B7-H3 and anti-CD22) and the hinge region. The specific binding of the respective TAA-overexpressing cells was verified for all constructs, and significantly improved binding properties were demonstrated in comparison with the native CPAb. The results of the binding and internalization assays also supported the hypothesis of predominantly TAA-mediated binding and endocytosis of the constructs. The investigation of the cytosol-penetrating properties of the bispecific constructs has led to the verification of the relevance of the endosomal furin cleavage site, as well as the tumor specificity of the constructs. It is noteworthy that constructs lacking prior cleavage of the masking unit were unable to penetrate the cytosol despite internalization, indicating that the inhibitory function of the mask is also preserved in the acidic environment of the endosomes. This study presents the first modular approach for TAA-specific intracellular transport of cargo proteins into tumor cells, simultaneously enabling cytosolic translocation conditioned upon activation in the TME. The third project focused on the investigation of tumor cell-specific cell penetration using an antibody-directed enzyme prodrug therapy (ADEPT) approach. The cell-penetrating peptide L17E was masked by the attachment of enzyme-cleavable acetyl protecting groups to the lysines of the peptide. The resulting inhibitory effect on cell penetration was then analyzed and the regeneration of cell penetration following incubation with the deacetylase SIRT2 was confirmed in different studies. To conditionally activate the masked peptide-cryptophycin conjugates in tumor cells, antibodies (trastuzumab and anti-B7-H3) were conjugated with SIRT2. Finally, the penetration of the cytosol with and without antibody-SIRT2 conjugate was tested in various cell lines through cell proliferation analysis. The studies demonstrated the feasibility of antibody-mediated peptide deacetylation as well as the potential for achieving conditionally activatable and TAA-specific cell penetration. The project thus represents the first approach for the SIRT2-dependent generation of conditionally activatable, cell-penetrating peptides using an ADEPT-like approach for TAA-specific cargo transport. Such as antibody fragments, peptides with a potential therapeutic effect can be sorted for affinity binders in a high-throughput process utilizing YSD in conjunction with FACS. In the case of highly structured peptides, such as cystine knots comprising three structure-determining disulfide bridges, a rational design of the peptide library is essential to obtain the three-dimensional structure and, consequently, the formation of the binding-relevant loops. In the last project, the methodology of YSD, as well as the subsequent peptide synthesis and binding studies, were described in detail.