Purpose To assess ranibizumab (Lucentis) penetration into the retina after topical administration in a rabbit model. Methods Ranibizumab was topically applied to the right eye of rabbits according to three regimens: every 2 h (q2hr), four times daily (qid), and twice daily (bid). Intraocular penetration of ranibizumab was assessed at 3, 7, 14, 21, and 28 days following initiation of drops. At each time point, the anterior chambers, vitreous cavities, and blood of one of the rabbits from each subgroup were sampled for ranibizumab detection using enzyme-linked immunosorbent assay (ELISA), and both eyes were then enucleated for ranibizumab detection in the retina by confocal immunohistochemistry (CI). Another group of rabbits received intravitreal ranibizumab and was similarly sampled for comparison. Results CI showed ranibizumab staining in the right retina after 7 and 14 days of q2hr topical administration in two out of four experiments. No ranibizumab was detected in the left retina at any of the sampling time points. ELISA was positive in the vitreous of the right eye at 14 and 21 days in the q2hr treated rabbits in one out of four experiments. No ranibizumab was detected in the qid and bid subgroups. CI and ELISA of the aqueous and vitreous were consistently positive in the intravitreal group. Mild ranibizumab levels were detected in the blood in both the topical and intravitreal groups. Conclusions Topically applied ranibizumab can be detected in the retina following high-frequency administration in a rabbit model. A trans-scleral route of penetration is suggested.