Pancreatic ductal adenocarcinoma (PDAC), with its devastating prognosis and limited treatment options, necessitates innovative therapeutic strategies. T-cell-based immunotherapy has shown promise for various cancers, including PDAC. Thus, a comprehensive understanding of the HLA-peptidome in PDAC is essential to pave the way for the effective design of immunotherapy and other interventions. In this study, we employed off-line high-pH reverse-phase (HPH-RP) fractionation of peptides extracted from immunoaffinity purified HLA class I molecules isolated from the surface of Panc1 cells in the absence and presence of cytokine stimulation. We demonstrate that this strategy is a relatively simple and rapid technique that significantly expands the depth of coverage of the PDAC immunopeptidome using data-dependent acquisition strategies and allowing the identification of more potentially actionable peptide antigens. Whilst cell lines provide an abundant source of material for immunopeptidomics analysis, clinical samples and biopsies from pancreatic cancer patients typically involve much smaller amounts of material. Thus, we performed a proof of concept study and demonstrated that immunopeptidome characterization was feasible from cell pellets of only 1 million cells (equivalent to approximately 1mg of tissue), using a spectral library generated from the HPH-RP fractionated material and the ZenoSWATH DIA-MS workflow on the SCIEX 7600 ZenoToF system. These studies form the basis for future clinical translational immunopeptidomics approaches to screen tumor antigen presentation in PDAC biopsies and provide new opportunities for immune-based therapies.