Modifications to surface-exposed residues of recombinant Adeno-associated virus (AAV), incorporation of novel regulatory elements into the vector cassette, alternative injection routes, and combinations thereof are being explored as strategies to enhance gene delivery to retina. We previously showed that intravitreal injection of a novel AAV capsid was capable of transducing all neuronal layers of the retina, including photoreceptors (PRs). We also demonstrated that this capsid, when combined with a novel promoter (“Ple155”) was capable of driving therapeutic nyctalopin (Nyx) expression exclusively in ON bipolar cells (ON BCs) and partially restoring retinal function to a mouse model of complete Congenital Stationary Night Blindness (CSNB1) following early (postnatal day 2) treatment. In order to achieve more efficient transduction of PRs and ON BCs, further modifications will be required. Methods with which to quantify relative transduction efficiencies of novel variants in vivo are warranted. The purpose of this study was to develop robust methods for quantifying transduction efficiency or transgene expression in PRs and ON BCs, respectively by rAAVs in vivo. Specifically, we sought to develop methods that don't require time consuming subjective analysis. To do so, we employed two transgenic mouse models: Nrl-GFP and Grm6-GFP mice which have constitutively fluorescent rod PRs and ON BCs, respectively. Adult mice (~postnatal day 30) were injected either subretinally (SR) or intravitreally (Ivt) with rAAVs containing the mCherry transgene. Fundoscopy was performed at 1 month post injection. Immediately thereafter, retinas were dissociated and evaluated by FACS for GFP and mCherry fluorescence, the overlap of which indicated targeted transduction of either ON BCs or rods, depending on mouse model. A subset of retinas was also evaluated with immunohistochemistry. In agreement with previous experiments, Ivt and SR delivered AAV2(quadY-F+T-V) and AAV2(4pMut) transduced a relatively large percentage of retina, including PRs. However, ON BC transduction was minimal, with ~6% transduced by Ivt AAV2(quadY-F+T-V)-CBA-mCherry delivery. AAV2 weakly transduced retina via both delivery routes. AAV2(4pMut)ΔHS, which lacks canonical HSPG binding residues, failed to transduce retina by Ivt injection, but efficiently transduced rods (75%) following SR delivery, indicative of significant ‘lateral spread’ from the injection bleb. Incorporation of the ON BC-specific promoter (“Ple155”) used in the CSNB1 gene replacement study abolished non-ON BC retinal expression. Experiments are ongoing to assess the extent to which rAAVs containing Ple155 can increase expression efficiency in ON BCs relative to CBA, and to determine whether the addition of other transcriptional and post transcriptional regulatory elements can further increase rAAV-mediated expression in these cells. In summary, we have developed a method for quantifying transduction of PRs and ON BCs by novel rAAVs in vivo. As the AAV vector toolkit expands, such a method will be highly useful for scoring relative performance of novel vectors. We also conclude that while the ubiquitous CBA promoter effectively drives transgene expression in PRs, it drives relatively poor transgene expression in ON BCs and caution that CBA should not be considered as a default universal promoter for retinal expression.