Synthetic peptides are used widely in research, including immunology and vaccine development. In particular, peptides can be included as antigens in vaccines or restimulation of T cells in ELISpot experiments. Purification of peptides using automated flash chromatography or HPLC can commonly yield greater than 99% purity, however the use of these columns and glassware for multiple peptides provides an opportunity for cross-contamination. This low-level contamination may not be detected by usual measures such as mass spectrometry but the sensitive nature of the immunological readout can be affected by these impurities. This can lead to identification of false-positives in antigen discovery. Here, we present a cross-contamination-free workflow for the preparation of peptides to be used in glycolipid-peptide conjugate vaccines. This includes single-use consumables for the process from synthesis of the peptide to purification of the final vaccine.