Updated publication reference for PubMed record(s): 33298915. The features of peptide antigens that contribute to their immunogenicity are poorly understood. Although the stability of peptide-MHC (pMHC) is known to be important, current assays assess this interaction only for peptides in isolation and not in the context of natural antigen processing and presentation2–4. Here, we present a novel method which provides a comprehensive and unbiased measure of pMHC stability for thousands of individual ligands detected simultaneously by mass spectrometry (MS). The method allows rapid assessment of intra- and inter-allelic differences in pMHC stability and reveals broader profiles of stability than previously appreciated. The additional dimensionality of the data facilitated the training of a model which improved the prediction of peptide immunogenicity, specifically of cancer neoepitopes. This assay can be applied to any cells bearing MHC or MHC-like molecules, offering insight into not only the endogenous immunopeptidome, but also that of neoepitopes and pathogen-derived sequences.