Purpose. To identify a novel, sensitive, nonradioactive leakage assay that can be used in the assessment of retinal vascular permeability in rats and mice. Methods. Breakdown of the vascular barrier was induced by vascular endothelial growth factor (VEGF), lipopolysaccharide (LPS), or diabetes. Biotinylated bovine serum albumin (bBSA) was administered as a tracer. After perfusion with lactated Ringer's solution, extravasated bBSA was detected with immunoprecipitation and Western blot analysis or sandwich ELISA. The results were then normalized against the final bBSA plasma concentration, the circulation time, and the protein concentration of the tissue. Results. Six hours after VEGF injection, BRB breakdown was quantified in the injected eye and was 2.5-fold higher than in the contralateral phosphate-buffered saline (PBS)-injected eye (n = 6 rats, P < 0.01). Intravitreal LPS injection induced severe inflammation in the directly injected eye and moderate inflammation in the contralateral untreated eye. Leakage was six- and threefold higher, respectively, compared with that in the untreated control animals (n = 5 rats, P < 0.01). Nine-month diabetic rats had a threefold increase in vascular leakage compared with age-matched control animals (n = 6 retinas, P < 0.05). Twenty-four hours after intraperitoneal administration of LPS in mice, the animals showed increased vascular leakage in all tissue organs examined (retina, 1.7-fold; brain, 1.5-fold; and kidney, 1.3-fold). Conclusions. bBSA can serve as an effective alternative to the current methods used for quantitating vascular leakage and especially the blood-retinal barrier breakdown. It is reasonably easy to perform, low in cost, and adaptable to experiments in mice.