The HLA-I peptide (pHLA-I) pathway leads to the presentation of peptides derived from endogenous antigens (including cancer-associated antigens) to T-cells for immunosurveillance. Collectively, these cell surface-presented peptides make up the cell’s immunopeptidome. Although they make good candidates for CD8+ T-cell immunotherapy, clinical trials with peptide vaccines have shown poor overall response rates. This creates an urgent need to expand the repertoire of novel peptide antigen targets. To broaden this repertoire, we used a previously published bioinformatic tool (Hybrid Finder) to analyse the immunopeptidome and detect spliced peptides. Spliced peptides are formed by a transpeptidation reaction occurring in the proteasome, and studies have shown that they could represent up to 30% of the immunopeptidome. They are an attractive inclusion in peptide-based vaccines as they broaden the repertoire of available targets and studies have shown that these peptides are immunogenic in nature. Whilst peptide splicing has been attributed to proteasomal-catalysed processes, it is unknown what effect other components of the antigen processing and presentation machinery (APPM) play. In this study, we used a combination of a panel of APPM knockout cells as well as inhibitors of various components of the APPM. Some initial immunopeptidomics analyses of a melanoma cell line treated in the presence or absence of an inhibitor of ERAP, an APPM component, showed that there was no significant change in the proportion and abundance of spliced peptides presented between different treatments. Since there was no abrogation in spliced peptide presentation, this suggests that other components could impact spliced peptide presentation. We anticipate that the knockout of TAP or components of the proteasomal complex would abrogate spliced peptide presentation thus conclusively explaining splicing.