The neonatal mouse retinal vascularization model has been widely used in the vascular biology field to investigate mechanisms of angiogenesis and arterial-venous fate specification during blood vessel formation and maturation. Recent advances in next-generation sequencing can further elucidate mechanisms of blood vessel formation and remodeling in this, as well as other, vascular development models. However, an optimized method for isolating retinal endothelial cells that limits tissue digestion-induced cell damage is required for next-generation sequencing applications. In this study, we established a method for isolating neonatal retinal endothelial cells that optimizes cell viability and purity. The CD31+/CD45− endothelial cell population was fluorescence-activated cell sorting (FACS)-isolated from digested postnatal retinas, found to be highly enriched for endothelial cell gene expression, and exhibited no change in viability for 60 min post-FACS. Thus, this method for retinal endothelial cell isolation is compatible with next-generation sequencing applications. Combining this isolation method with next-generation sequencing will enable further delineation of mechanisms underlying vascular development and maturation.