AAV capsid viral proteins (VPs) are important constituents of AAV product and play an important rule for immunogenicity and tissue tropism in gene therapy. Fully characterization of viral proteins is required to ensure the safety, quality and efficacy of AAV products. Conventional AAV capsid protein characterization methods such as ELISA, SDS-PAGE are time consuming and do not provide direct viral capsids identification information. HPLC-MS technology enables faster, more sensitive and more quantitative approaches for capsid protein characterization and is emerging as a fast alternative analytical method for AAV capsid protein characterization. However, unlike the therapeutic proteins, the AAV sample is more complex in structure and limited in sample volume due to small batch sizes. Plus, the titer of AAV products are generally Low, E11 – low E13 vg/mL range, yielding low capsid protein concentrations. The adapted mass spectrometer needs to have high sensitivity and high resolution to detect low abundant VPs and associated truncated forms while keeping great mass accuracy. In this talk, we will demonstrate that the high sensitivity and ultra high resolution offered by Thermo Scientific^TM Orbitrap Exploris^TM 480 mass spectrometer enables rapid characterization of AAV6 capsid proteins through intact, top-down and peptide mapping analysis with great sensitivity and mass accuracy. The average masses of VPs with or without modifications were determined with less than 3 ppm mass accuracy even down to 28 ng level using intact mass analysis. The sequences of detected VP3 truncated forms were confidently confirmed with the top-down analysis. By using pepsin enzyme for digesting the VPs, 100% sequence coverage of AAV6 VP1 and site specific PTM identification/ relative quantification were achieved in a single HPLC-MS run using peptide mapping analysis.