Adeno-associated virus (AAV) vectors are the principal vehicle for gene therapy. In a large share of humans, immunity against AAVs can reduce or completely inhibit transgene expression, and can lead to adverse outcomes. The level of immunity can be estimated by assaying AAV neutralization level of the blood serum. Surprisingly, there is no widely adopted sensitive assay for the quantification of AAV neutralization.Results: We developed an in vitro neutralization assay, and quantified its reproducibility and sensitivity via longitudinal evaluation of neutralization in preclinical models and humans. Compared to established protocols, our method exhibits lower variability and reports stronger neutralization levels when tested on the same serum samples.Conclusion: Our results may offer readout modality of neutralization with superior accuracy and reproducibility, thereby serving as a cross-species validated foundation for safety assessment of AAV constructs.Supported by ELKH-POC-2021-026 grant, the Lendület (“Momentum") Programme of the Hungarian Academy of Sciences, NKFIH FK18 No. 129120 for DH. Co-operative Doctoral Program from Ministry of Innovation and Technology of Hungary (NKFIH) for BK. NKFIH OTKA 134799 for MB.