Mutations in the human BEST1 gene are responsible for a number of distinct retinal disorders known as 'bestrophinopathies', for which there are no current treatments. The protein product, bestrophin-1, is expressed in the retinal pigment epithelium (RPE) where it localises to the basolateral membrane and acts as a Ca2+-activated chloride channel. Recent studies have shown successful BEST1-mediated gene transfer to the RPE, indicating human clinical trials of BEST1 gene therapy may be on the horizon. A critical aspect of such trials is the ability to assess the efficacy of vector prior to patient administration. Here, we present an assay that enables the quantitative assessment of AAV-mediated BEST1 chloride conductance as a measure of vector efficacy. Expression of BEST1 following transduction of HEK293 cells with AAV.BEST1 vectors was confirmed by liquid chromatography, western blot and immunocytochemistry. Whole-cell patch-clamp showed increased chloride conductance in BEST1-transduced cells compared to sham-transduced and untransduced controls. Exogenous chloride current correlated to BEST1 expression level, with an enhanced AAV.BEST1.WPRE vector providing higher expression levels of BEST1 and increased in chloride conductance. This study presents in vitro electrophysical quantification of bestrophin-1 following AAV-mediated gene transfer, providing vital functional data on an AAV gene therapy product which will support a future application for regulatory approval.