<p>Millions of people worldwide suffer from vision impairments, significantly affecting their quality of life and mental health and imposing a massive economic burden on healthcare. The eye, converting light into signals for the brain, plays a key role in vision, with the retina being especially vital yet susceptible to damage. Gene therapy holds promise for effectively treating vision-related diseases. However, in basic research, screening for effective gene carriers often lacks quantitative evaluation. One major challenge in translating promising nanoparticles from in vitro studies to (pre)clinical applications is the presence of retinal drug delivery barriers. Ex vivo experimentation could help overcome that by providing insights into these barriers early on, yet much of the current research relies heavily on qualitative assessments. Specifically, the lack of quantitative methods to assess nanoparticle penetration in the retina moved us to develop a flow cytometry-based method for large animal retinal tissue. Our method proved to efficiently quantify both nanoparticle uptake and transfection in the retina, corroborating our qualitative outcomes and positioning itself as a useful tool to support the drug and gene delivery community.</p> Graphical Abstract <p></p>