Peptides presented by Human Leukocyte antigen (HLA) class I and II molecules form an important component of the adaptive immune response against viruses, bacteria and tumours. Identifying HLA-bound peptides is therefore crucial to understand the specificity of T cell responses in cancer and infectious disease. Due to sample limitations, it is often difficult or impossible to confirm epitope presentation in clinical biopsy material. To develop truly personal medicine, target epitopes need to be directly identified or validated in patient tumours at the peptide level. We have developed a microscale HLA immunoprecipitation protocol which when combined with a tandem mass tag (TMT)-based barcoding approach was able to identify HLA-bound peptides from just 1000 cells. We demonstrate the clinical utility of this approach by confirming the presentation of around 1000 peptides from a challenging melanoma biopsy (~1-20mg in total), including the detection of a potential neoepitope and several known melanoma epitopes.