Penicillin hypersensitivity is recognised as a major public health concern with up to 11.5% of recipients reporting adverse reactions. Traditionally, penicillin allergies have been considered to be primarily IgE-mediated, however, there is emerging evidence for a key role for T cell-mediated reactions. Possessing the ability to form covalent bonds (known as haptenation) with serum proteins, penicillins create neo-antigens that have the potential to be immunogenic. Upon processing and presentation by antigen-presenting cells, these haptenated neo-antigens may activate drug-responsive T cells, leading to a range of cutaneous adverse drug reactions from mild rashes to more severe pathologies involving organ damage such as drug-induced liver injury. A group of 10 patients were recruited from The Alfred Hospital Allergy Clinic based on their history of adverse drug reaction to penicillins. Of these patients, only 40% tested positive for serum IgE against the causative drug, suggesting that other factors/cellular populations (i.e. T cells) contribute to the adverse reaction. Here we focus on one hypersensitive patient, who exhibited drug-responsive T cells activated via the human leukocyte antigen (HLA) class-I molecule HLA-A*02:01. We hypothesised that this common Caucasian HLA allotype presented a haptenated neo-antigen to T cells responsible for the ensuing drug reaction. Using an immunopurification LC-MS/MS workflow, we explored the HLA-A*02:01 immunopeptidome isolated from patient-derived B-cells treated with or without penicillin. Utilising haptenated human serum albumin, we defined a set of robust criteria for identification of penicillin-haptenated peptides, which when applied to this data set showed evidence for three HLA-A*02:01 bound penicillin-haptenated peptides. Together, these results set the basis for further characterisation of haptenated peptides in other HLA-A*02:01+ penicillin hypersensitive patients and will provide novel molecular insights into this common drug reaction.