Updated project metadata. T cell recognition of human leukocyte antigen (HLA)-presented tumor-associated peptides is central for cancer immune surveillance. Mass spectrometry (MS)-based immunopeptidomics represents the only unbiased method for directly identifying and characterizing naturally presented tumor-associated peptides, which represent a key prerequisite for the development of T cell-based immunotherapies. This study reports on the de novo implementation of ion mobility separation-based timsTOF MS for next-generation immunopeptidomics, enabling high-speed and sensitive detection of HLA peptides. A direct comparison of timsTOF-based with state-of-the-art immunopeptidomics using orbitrap technology showed significantly increased HLA peptide identifications from benign and malignant primary samples of solid tissue and hematological origin. First application of timsTOF immunopeptidomics for tumor antigen discovery enabled (i) the expansion of benign reference immunopeptidome databases with more than 150,000 HLA peptides from 94 primary benign tissue samples, (ii) the refinement of previously described tumor antigens, and (iii) the identification of a vast array of novel tumor antigens comprising low abundant neoepitopes that might serve as targets for future cancer immunotherapy development.