To understand and treat immunology-related diseases, a comprehensive, unbiased characterization of major histocompatibility complex (MHC) peptide ligands is of key importance. Preceding the analysis by mass spectrometry, MHC peptide ligands are typically isolated by MHC immunoaffinity chromatography (MHC-IAC). Less often, mild acid elution (MAE) is used to extract MHC class I peptide ligands. MAE may provide a cheap alternative to MHC-IAC for suspension cells, but it is thought to be hampered by the high number of contaminating peptides not derived from the MHC. Here, we optimized the MAE protocol yielding MHC peptide ligand purities of more than 80%. We discovered distinct cysteinylation frequencies at individual positions of MHC peptide ligands and propose that MAE conserves the native cysteinylation pattern of MHC peptide ligands better than MHC-IAC. Our improved and carefully documented MAE workflow represents a high-quality, cost-effective alternative to MHC-IAC for suspension cells and should be applicable also in laboratories not specialized in MHC peptide ligand analyses.