Short peptides derived from cellular proteins may escape complete destruction during protein catabolism and finally serve as a showcase in the immune system. Exposed at the cell surface to scrutiny by T cells, MHC:peptide complexes mediate a highly specific and immediate information transfer from diseased cells to the cellular immune system. Numerous clinical vaccination trials have been carried out employing MHC-presented peptides for T-cell activation with encouraging results but so far without a final breakthrough. In this review, we briefly highlight the molecular basis of MHC-peptide interactions governed by specificity pockets and anchor residues, as summarized in allele-specific peptide motifs. State-of-the-art technology is comprehensively presented and gives an overview of modern mass spectrometric strategies used for qualitative and quantitative analysis of MHC ligands. We describe the details of the HLA-B*3801 peptide motif by comparing features of natural MHC ligands, resulting in a scoring matrix that enables epitope prediction from any viral or tumor antigen. The pronounced individuality in peptide presentation by MHC molecules, as reflected in the highly specific peptide motifs of different MHC allotypes or the tissue-specific MHC ligandomes, represents a current area of interest within this field. Finally, the identification of post-translational modifications--most important phosphorylations--and the promises this holds will be discussed in this chapter.