Immunotherapies provide effective treatments for previously untreatable tumors and identifying tumor-specific epitopes can help elucidate the molecular determinants of therapy response. Here, we describe a pipeline, ISOTOPE (ISOform-guided prediction of epiTOPEs In Cancer), for the comprehensive identification of tumor-specific splicing-derived epitopes. Using RNA sequencing and mass spectrometry for MHC-I associated proteins, ISOTOPE identified neoepitopes from tumor-specific splicing events that are potentially presented by MHC-I complexes. Analysis of multiple samples indicates that splicing alterations may affect the production of self-epitopes and generate more candidate neoepitopes than somatic mutations. Although there was no difference in the number of splicing-derived neoepitopes between responders and non-responders to immune therapy, higher MHC-I binding affinity was associated with a positive response. Our analyses highlight the diversity of the immunogenic impacts of tumor-specific splicing alterations and the importance of studying splicing alterations to fully characterize tumors in the context of immunotherapies. ISOTOPE is available athttps://github.com/comprna/ISOTOPE.