Subretinal injection is widely used in gene therapy research on age-related macular degeneration and retinitis pigmentosa. We investigated a method of injection and the maximum feasible volume for subretinal injections in rats and monkeys to support future studies for evaluation of new therapies in these animals. Physiological saline was injected subretinally into the eyes of male adult rats (n = 3 eyes/group) and cynomolgus monkeys (n = 3 eyes/group). The eyes were then examined by ophthalmoscopy (slit lamp and ocular fundus examinations), optical coherence tomography (OCT), intraocular pressure (IOP) measurements, or electroretinography (ERG). Rats received physiological saline at 1, 2, 5 µL/eye (2 sec/eye, bolus) and 5 µL/eye (5 µL/min). Monkeys received physiological saline at 50, 100, 150, 200, and 250 µL/eye (10 µL/sec). In all rats and cynomolgus monkeys, successful injection was visually confirmed by bleb formation using OCT at the appropriate sites immediately after injection. IOP increased more than 2-fold after injection of 5 µL/eye (5 µL/2 sec) compared with before injection but remained virtually unchanged at other volumes. Monkeys that received 200 or 250 µL/eye showed a marked increase in IOP immediately after injection, with functional abnormality observed in ERG. In conclusion, bleb formation at the appropriate sites was confirmed by OCT, demonstrating successful subretinal injections to rat and monkey eyes. When physiological saline solution is subretinally injected, the maximum feasible volume is considered to be 5 µL/eye (5 µL/min) in rats and 150 µL/eye (10 µL/sec) in cynomolgus monkeys, based on IOP and ERG results.