[15:18] Toole, Estee Adeno-Associated Viruses (AAVs) comprise an area of rapidly growing interest due to their ability to act as a gene delivery vehicle in novel gene therapy strategies and vaccine development. Peptide mapping is a common technique in the biopharmaceutical industry to confirm the correct sequence, product purity, PTMs, and stability. However, conventional peptide mapping is time-consuming and has proven difficult to reproduce with viral capsids because of their high structural stability and the suboptimal localization of trypsin cleavage sites in the AAV protein sequences. In this study, we present an optimized workflow that provides thorough characterization within one day. This workflow is also highly reproducible due to its simplicity having very few steps, and easy to perform proteolytic digestion utilizing thermally-stable pepsin, active at 70°C in acidic conditions. The acidic conditions of the peptic digestions drive viral capsid denaturation and improve cleavage site accessibility. We characterized the efficiency and ease of digestion through peptide mapping of the AAV2 viral capsid protein. Using nanoflow liquid chromatography coupled with tandem mass spectrometry, we achieved 100% sequence coverage of the low abundance VP1 capsid protein with a digestion process taking only 10 minutes to prepare and 30 minutes to complete the digestion.