Mass spectrometry (MS)-based methods coupled to reverse phase chromatography separation are a useful technology to analyze complex peptide pools that are comprised of different peptides with unrelated sequences. In antigen presentation, proteasomes generate a set of short peptides that are closely related and overlapping and in some instances may even have identical retention times and identical masses. In these situations, micro-liquid chromatography-MS/MS focused on each theoretical parent ion followed by manual interpretation optimizes the identification of generated peptides. The results suggest that the degradation of short antigens by the proteasome occurs by sequential cleavage.