Pre-existing neutralising antibodies (NABs) to Adeno Associated Virus (AAV) can prevent the success of gene therapy. A variety of assay have been developed to detect NAB in patients considering gene therapy including a mouse based in vivo transduction inhibition assay for AAV serotype 8 which requires a high multiplicity of infection (MOI) for successful in vitro gene transfer.  The in vivo assay has several disadvantages, namely, the requirement for mice, cost and the long 4-6 week timeline associated with ordering and acclimatising the mice, as well as the in vivo phase. We have therefore developed a rapid in vitro assay that enables screening for NABs with a turn-around time of 2 days.  Initially CMV NanoLuc and a secreted version packaged into AAV3, 5 and 8 were compared over a range of MOIs from 0.1-1000 on HEK 293T cells and incubation time from 6-24hrs. NanoLuc was selected with an MOI of 1 for AAV3 and 100 for AAV5 and 8 with a 24hr incubation time.  We evaluated 38 plasma samples from healthy controls, the median 50% neutralising dilution was 837, 123 and 44 for AAV3, 5 and 8 respectively, demonstrating the high sensitivity of the AAV3 assay. A third of plasma samples were negative for neutralising antibodies, to all three serotypes, only one sample was negative for AAV5, but positive for AAV3 and AAV8. There was also a good correlation with neutralising dilution and total antibody titre measured by ELISA.