The current “gold standard” for the identification of MHC-restricted peptides is conventional immunoprecipitation/mass spectrometry (MS); however, this approach requires a relatively large amount of sample, complex purification procedures, and computational analyses to assign peptides to individual alleles. Here, we provide instructions for the expression, purification, and MS identification of MHC-presented peptides of human and non-human, classical and non-classical, class I and class II proteins using a readily expressible, soluble, and easily purifiable single-chain dimer construct. This procedure enables the identification of up to tens of thousands of allele-specific peptides per run for peptidomics, ligandomics, therapeutic targeting, and motif analyses. Also included are instructions for construct design, streamlined lentiviral transfection and transduction, and computational methods for high-throughput processing of MS results, yielding high confidence peptide lists, motifs, and multiple analytics.