Human leukocyte antigen class I (HLA-I) molecules sample the proteome following the degradation of intracellular proteins via the proteasome and other proteolytic mechanisms. These complexes of HLA-I and peptide (pHLA) are then recognized by T cells and the nature of the bound peptide ligand is the key driver of adaptive immunity. We have developed a sophisticated data-driven workflow for the identification of distal and proximal cis-spliced as well as trans-spliced (where distinct proteins contribute peptide segments) peptide antigens. We have shown for 17 different monoallelic cell lines, that up to 40% of pHLA are generated through post-translational splicing mechanisms. We also found that spliced peptides display canonical HLA-binding sequence features (1). To understand the biological relevance of spliced peptides, we have interrogated the immunopeptidome of HLA-I molecules immunoaffinity purified from influenza-infected cells using LC-MS/MS and the novel bioinformatic pipeline. We have found around 42% of influenza derived p-HLAs (83 out of 198 peptides) are spliced peptides. We have also applied our approach to p-HLA derived from melanoma cell lines and identified 38766 peptides of which 6% were cis-spliced and 21% trans-spliced. Of note, more than more 100 spliced peptides were derived from melanoma-associated antigens (MAA) and ~40% of known MAA were only represented by spliced peptides. Immunogenicity studies of a subset of the MAA shown that multiple of the tested spliced peptides were highly immunogenic, compared to a smaller fraction of linear peptides. Our results highlight the complexity and diversity of HLA peptide antigen presentation and identify a mechanism whereby the available peptidome is diversified to enhance immunity. We found spliced peptides may yield more immunogenic epitopes than are available from the viral/cancer genome. Moreover, some antigens lack high-affinity HLA-ligands and the peptide splicing mechanism can generate higher affinity neoepitopes for interaction with host HLA allomorphs. Understanding the nature and abundance of spliced peptides has a high relevance for our understanding of potential novel targets of T cell immunity and will have significant implications for further immunotherapeutic approaches.