Neoepitopes are the only truly tumor-specific antigens. Although potential neoepitopes can be readily identified using genomics, the neoepitopes that mediate tumor rejection constitute a small minority, and there is little consensus on how to identify them. Here, for the first time to our knowledge, we use a combination of genomics, unbiased discovery mass spectrometry (MS) immunopeptidomics, and targeted MS to directly identify neoepitopes that elicit actual tumor rejection in mice. We report that MS-identified neoepitopes are an astonishingly rich source of tumor rejection-mediating neoepitopes (TRMNs). MS has also demonstrated unambiguously the presentation by MHC I, of confirmed tumor rejection neoepitopes that bind weakly to MHC I; this was done using DCs exogenously loaded with long peptides containing the weakly binding neoepitopes. Such weakly MHC I–binding neoepitopes are routinely excluded from analysis, and our demonstration of their presentation, and their activity in tumor rejection, reveals a broader universe of tumor-rejection neoepitopes than presently imagined. Modeling studies show that a mutation in the active neoepitope alters its conformation such that its T cell receptor–facing surface is substantially altered, increasing its exposed hydrophobicity. No such changes are observed in the inactive neoepitope. These results broaden our understanding of antigen presentation and help prioritize neoepitopes for personalized cancer immunotherapy.