Abstract There is a strong need for nanobodies targeting novel cancer-associated antigens to advance radioligand imaging and antibody-based therapeutics. In this study, we investigated whether non-targeted Llama immunization using tumor cells, combined with non-targeted phage display panning on human cell lines, could yield nanobodies specific to Prostate Specific Membrane Antigen (PSMA). Nanobody selection using both classical three round PSMA negative-positive panning and single round panning of cell lines positive or negative for PSMA showed clear enrichment for PSMA binders in both strategies. Using shRNA knockdown, flow cytometry, cell-ELISA, immunohistochemistry and structural modeling and docking, we confirmed the PSMA targeting of selected nanobodies. Two distinct epitopes were predicted to be bound by nanobodies PSMANb9 and A7 (JVZ-007), corroborated by epitope competition assays. These findings support the feasibility of non-targeted immunization and panning strategies for isolating clinically relevant cancer nanobodies.