This study characterised the array of benzylpenicillin-modified ligands within the immunopeptidome of HLA-A*02:01. We further describe a novel target of penicillin within the immunopeptidome and proteome. Identification of penicillin-modified ligands within the immunopeptidome will allow future immunogenicity assays to aid in understanding how T cells can mediate penicillin-induced hypersensitivities.