Human arginase-1 (hArg1) is a pivotal enzyme implicated in over 30 medical conditions, possessing significant therapeutic potential, particularly in cancer treatment and metabolic disorders. Engineering hArg1 to enhance its kinetic and immunologic properties is crucial for its clinical application. Therefore, the development of robust, high-throughput methods for the evolution and engineering of hArg1 brings vital advancements to the field. This thesis aims to expand the toolkit for studying and engineering hArg1. Chapter 1 provides a general introduction, highlighting the role and importance of therapeutic enzymes in treating various diseases. It reviews the use of enzyme cascades for directed evolution of biocatalysts and underscores the importance of throughput in such screenings. It also addresses the challenges in enzyme therapeutics, such as immunogenicity, and highlights the need for advanced engineering techniques. Chapter 2 details the development of a novel enzyme cascade assay for high-throughput screening of hArg1 activity. This innovative one-pot assay combines hArg1 with ornithine decarboxylase, putrescine oxidase and horseradish peroxidase to generate a colorimetric or fluorescent signal, facilitating continuous kinetic readouts. The assay was used to screen a curated library of hArg1 variants as a proof-of-concept, identifying the R21E variant with enhanced catalytic turnover. This robust assay system is adaptable for directed evolution, significantly advancing the toolkit for hArg1 engineering. Chapter 3 includes the first-ever integration of genetic code expansion with protein bacterial surface display, which was implemented to incorporate non-canonical amino acids (ncAAs) into hArg1. More specifically, S-allylcysteine (SAC) was incorporated at amber stop codons using various orthogonal aminoacyl-tRNA synthetases. Methanococcoides burtonii pyrrolysyl-tRNA synthetase was selected as the best system for incorporating SAC into displayed hArg1. The study introduces an amber scanning technique by systematically introducing amber stop codons across hArg1 and assessing them by integrating high-throughput screening and next-generation sequencing. As a result, ncAA incorporation efficiency per residue was mapped across hArg1. The findings provide detailed insights into the structural and sequence dependency of hArg1 for SAC incorporation. Furthermore, site-specific bioconjugation was achieved by targeting SAC and introducing mutually bioorthogonal chemistries. Bioconjugation efficiency was assessed with an amber scanning method, which revealed further structural and dynamic insights of hArg1. This tool served as a novel method for epitope mapping by integrating site-specific bioorthogonal PEGylation and immunofluorescence assays. This approach leverages site-specific PEGylation to sterically hinder antibody binding, providing high-resolution data on the impact of PEGylation on immune recognition and facilitating the identification of 4 epitopes. This innovative technique produces information on optimal PEGylation strategies, advancing towards the development of more effective and less immunogenic therapeutic enzymes. The thesis concludes with Chapter 4 summarizing the advancements made in developing tools for engineering hArg1 and emphasizing the significance of these tools in improving therapeutic relevance and reducing immunogenicity. The research highlights the potential for further applications of these methodologies in broader contexts, envisioning the integration of high-throughput activity assays with advanced amber scanning techniques to address current challenges in enzyme therapy. This body of work significantly contributes to the field of protein engineering, providing novel tools and methodologies that enhance our ability to develop and optimize therapeutic enzymes, particularly human arginase-1.