Major histocompatibility complex (MHC) class I molecules present short peptide ligands on the cell surface for interrogation by cytotoxic CD8(+) T cells. MHC class I complexes presenting tumor-associated peptides such as neoantigens represent key targets of cancer immunotherapy approaches currently in development, making them important for efficacy and safety screenings. Without peptide ligand, MHC class I complexes are unstable and decay quickly, making the production of soluble monomers for analytical purposes labor intensive. We have developed a disulfide-stabilized HLA-A*02:01 molecule that is stable without peptide but can form peptide-MHC complexes (pMHCs) with ligands of choice in a one-step loading procedure. We illustrate the similarity between the engineered mutant and the wild-type molecule with respect to affinity of wild-type or affinity-matured T cell receptors (TCRs) and present a crystal structure corroborating the binding kinetics measurements. In addition, we demonstrate a high-throughput binding kinetics measurement platform to analyze the binding characteristics of bispecific TCR (bsTCR) molecules against diverse pMHC libraries produced with the disulfide-stabilized HLA-A*02:01 molecule. We show that bsTCR affinities for pMHCs are indicative of in vitro function and generate a bsTCR binding motif to identify potential off-target interactions in the human proteome. These findings showcase the potential of the platform and the engineered HLA-A*02:01 molecule in the emerging field of pMHC-targeting biologics.