Immunopeptidomics relies on LC-MS/MS for comprehensive profiling of HLA-bound peptides, yet detection remains challenging due to their non-tryptic nature, variable lengths, and lack of basic residues, which limit ionisation and fragmentation efficiency. To address these limitations, we investigated the impact of incorporating 5% dimethyl sulfoxide (DMSO) into LC-MS/MS mobile phase buffers on immunopeptidomic workflows. Using B-lymphoblastoid cell lines expressing HLA class I and II alleles and elastase-digested HeLa lysates as a surrogate for non-tryptic peptides, we assessed peptide identification, ionisation efficiency, charge state distribution, and fragmentation quality. DMSO significantly increased peptide identifications across all sample types, with gains of ~1.33-fold for HLA class I, ~1.55-fold for HLA class II, and ~1.24-fold for elastase digests. Improvements were systematic and reproducible, driven by enhanced electrospray ionisation, higher charge states, and superior MS2 spectral quality, evidenced by a two-fold increase in b- and y-ion intensities. Importantly, DMSO did not introduce major sequence bias, preserving motif integrity and predicted binding characteristics. Overall, these findings establish DMSO as a robust additive for improving sensitivity and reliability in immunopeptidomics, particularly for low-input or clinically derived samples.