Recent data indicate that endogenous mutated cancer proteins can be processed and presented as HLA binding peptides, leading to their recognition in vivo as "non-self." Targeting such neoantigens would enable immune cells to distinguish between normal and cancerous cells, avoiding the risk of autoimmunity. So far, discovery of such neoantigens relies mainly on prediction-based interrogation of the "mutanome" using genomic information as input, followed by highly laborious and time-consuming T cell screening assays. Currently, mass spectrometry is the only unbiased methodology to comprehensively interrogate the naturally presented repertoire of HLA binding peptides, including peptides derived from tumor-associated antigens and post-translational modified peptides. This chapter describes a detailed protocol for in-depth and accurate mass spectrometry based immunopeptidomics, enabling the direct identification of tissue-derived neoantigens extracted from human tumors.