Endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1, ERAP2), along with Insulin-Regulated Aminopeptidase (IRAP), play key roles in antigen processing by trimming precursor peptides for major histocompatibility complex (MHC) class I presentation, thereby contributing to adaptive immune regulation. Dysregulation of these enzymes has been implicated in various diseases, including autoimmune disorders and cancer. This chapter provides a comprehensive, step-by-step methodological guide for the expression, purification, and functional characterization of recombinant ERAP1, ERAP2, and IRAP. Detailed protocols include baculovirus-mediated expression in insect cells, affinity chromatography for protein purification, and a suite of functional assays to study enzymatic activity, substrate specificity, and inhibitor screening. Fluorogenic, colorimetric, and HPLC-based assays are described, offering versatile tools for kinetic analysis and inhibitor evaluation. These protocols are designed to yield high-purity, functionally active enzymes suitable for biochemical, structural, mechanistic, and drug discovery studies.