Despite existing vaccine strategies, influenza viruses inflict a high level of mortality and morbidity worldwide and are a significant economic burden. Current vaccine formulations induce predominantly antibody-based immune responses against the surface hemagglutinin (HA) which can differ greatly between strains resulting in limited cross-strain protection. CD8 T cells recognise peptidic antigens generated through the breakdown of viral proteins within infected cells and displayed by the HLA class I molecules (HLA-I) at the cell surface. Thus, they have the capacity to initiate responses against more conserved internal viral proteins and therefore broad recognition across strains. Rational vaccine formulation requires knowledge of which viral components instigate immune responses. Therefore, we have utilised a mass spectrometry-based epitope discovery program, interrogating the immunopeptidome (array of peptides presented by the HLA) of diverse HLA molecules to define the virus-derived peptides available for immune surveillance. Empowered by an in-house developed workflow that combines data-driven de novo sequencing (PEAKS 8.5) with proteome database searching, we have mapped the contribution of both genome templated (linear) and a newly recognised subset of non-genome templated (spliced) peptides. Current work moves to assess the contribution of both linear and spliced peptide subsets to the anti-influenza immune response in healthy donors. Not only are these data sets helping define the rules for peptide splicing, but they will inform the design of vaccine strategies that maximise T cell responses and the progress towards a universal influenza vaccine.