The large-scale identification of MHC class I presented peptides is indispensable for gaining insight into the fundamental rules of immune recognition as well as it is an invaluable tool in identifying potential targets for the immunotherapy of disease. In this chapter we briefly review the existing strategies for the analysis of MHC ligandomes and provide an in-depth protocol for the immunoaffinity purification of MHC class I presented peptides from primary tissues or cells.