Background: Immunopeptidomics is the MS/MS-based study of HLA-bound peptides. These HLA-peptide complexes, presented on cell surface, allow T-cells to monitor cellular protein expression. Immunotherapy’s goal is to direct T-cell responses to tumour-specific peptides, which often bear cancerrelated mutations or aberrant phosphorylation arising from oncogenic signaling events. As the peptide (rather than protein) is the species of interest, MS/MS-quality is paramount; however, collision-induced dissociation (CID) often fails to provide unambiguous identification of a subset of peptides. This may arise from phosphate loss during fragmentation, which hinders accurate identification and PTMlocalization, or lack of delineation between isobaric residues. Indeed, the prevalence of Ile/Leu sequence variants in the immunopeptidome has rarely been fully determined using MS/MS-based approaches. Thus, we leveraged a combination of Electron-Activated-Dissociation (EAD) and CID (ExCID) to generate hybrid MS/MS data to characterize immunopeptides. Methods: We isolated immunopeptides of the lymphoblastoid C1R (HLA-I and II) and pancreatic cancer Panc1 (HLA-I) cell-lines, and a portion of each were enriched for phosphopeptides using Zr-IMAC. MS/MS spectra were acquired using ExCID method with a KE=6eV for 20ms and dynamic collision energy. Results: HLA-peptides identified with either fragmentation modes (ExCID or CID) demonstrated similar sequence characteristics. In unenriched samples, across comparison groups, we observed 59-67% similar identification between ExCID and CID; 13.5-14.5% of peptides in each comparison were unique to ExCID. Importantly, ExCID generated diagnostic ions that allowed Ile/Leu-variants to be distinguished. Furthermore, ExCID outperformed CID for the identification and characterization of phosphorylated immunopeptides. Overall, we detected 547 Zr-IMAC-enriched phosphoimmunopeptides from 508 phosphosites. Additionally, we found isomeric phosphopeptides; while CID was unable to differentiate these phospho-isomers, ExCID provides spectral evidence for site-localization, corroborated with synthetic peptides. Conclusion: ExCID improves MS/MS fragment coverage and PTM site-localization and enables distinction of Ile/Leu variants, improving accuracy of immunopeptide annotation, paramount for selection of peptides for downstream immunotherapeutic applications.