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Pre-existing antibodies to viral capsids may have a negative impact on the efficacy and safety of adeno-associated virus (AAV)-based gene therapies. Total antibody (TAb) and/or cell-based transduction inhibition (TI) assays have been used to exclude seropositive individuals in clinical studies. Published AAV seroprevalence and patient enrollment criteria regarding antibody status lack comparability between assay formats, hindering a direct cross-study comparison. To identify critical factors impacting TI assay detection of AAV neutralizing antibodies (NAbs), we created a reporter construct expressing NanoLuc® luciferase (Nluc) that enabled a more sensitive and robust detection of AAV6 NAbs than using firefly luciferase. Assessment of additional factors including multiplicity of infection, cell lines, viral production, and capsid purity revealed the reporter is the major determinant of assay sensitivity impacting NAb detection. The Nluc reporter was further used to assess seroprevalence to AAV5, 8, and 9. Last, we compared AAV6 Nluc TI with two TAb assay formats. A higher correlation of Nluc TI was observed with direct binding (90%) than with the more sensitive bridging TAb assay (65%), suggesting both assay sensitivity and TAb formats contribute to AAV seropositivity concordance. Our results support a need to standardize assay formats to ensure proper assessment of pre-existing AAV immunity.