The interrogation of cell surface-presented immunogenic epitopes is of great importance to differentiate diseased cells in consequence to malignant transformation or viral infections. On the basis of this knowledge, next-generation immunotherapies against cancers, autoimmunity, or infectious diseases can be developed. The identification of altered peptide repertoires of transformed cells renders mass spectrometry-based analysis indispensable. This is evident considering the low correlation of gene or protein expression alterations, respectively, with changes in the peptide repertoire rendering those analyses less informative. Nevertheless, immunogenicity of peptides appearing to be exclusively found on diseased cells has to be finally proven in T cell-based assays. This review highlights the capabilities and limitations of mass spectrometry in the identification of entire immunopeptidomes, as well as individual potential immunogenic epitopes with a strong focus on cancer. Furthermore, an overview of state-of-the-art immunogenicity screens is presented.