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Adeno-associated virus (AAV) vector applications are often limited by capsid-directed humoral immune responses, mainly through neutralizing antibodies (NAbs), which are present throughout the human population due to natural AAV infections. Currently, antibody levels are often quantified via ELISA-based protocols or by cellular NAb assays and less frequently by in vivo NAb assays in mice. These methods need optimization for each serotype and are often not applicable to AAV variants with poor in vitro transduction. To tackle these limitations, we have established Meso Scale Discovery (MSD)-based assays for the quantification of binding antibodies (BAbs) and NAbs against the three most commonly used AAV serotypes, AAV2, AAV8, and AAV9. Both assays detect anti-AAV-IgG 1-3 with high sensitivity and consistency as shown in a screen of sera from 40 healthy human donors. Subsequently, BAb and NAb titers were determined for identification of seronegative animals in a non-human primate (NHP) cohort. Moreover, the MSD-based BAb assay protocol was extended to a panel of 14 different AAV serotypes. In summary, our platform allows a rapid and quantitative assessment of the immunological properties of any natural or engineered AAV variant irrespective of transduction efficiency and enables high-throughput screens., Competing Interests: All authors listed are employees of Boehringer Ingelheim Pharma GmbH & Co. KG or Boehringer Ingelheim Venture Fund GmbH., (© 2022 The Author(s).)