The production of Major Histocompatibility Complex (MHC) Class I proteins bound to different peptides of interest is essential for many immunological studies. However, the preparation of MHC-I molecules, whether wild-type or engineered, requires in vitro refolding with a high-affinity peptide, which stabilizes the complex, leading to a sufficient protein yield. This presents a challenge for subsequent peptide exchange reactions, commonly used to generate pMHC-I tetramer libraries. Here, we describe an optimized protocol for chaperone-mediated exchange of a high-affinity placeholder peptide for an array of target peptides. Using this protocol, a pMHC-I tetramer library encompassing 135 different antigen specificities can be produced within 3 days.