Dexamethasone is a corticosteroid with proven efficacy for treating both anterior- and posterior-segment ocular diseases. Delivery of drugs to the back of the eye has always been a challenge, with dexamethasone being no exception. There are multiple delivery routes to the retina, with each exhibiting different pharmacokinetics, depending on the drug molecule and specific route of administration. In this study, we used intravenous (IV), subconjunctival (SC), and intravitreal (IVT) injections in rabbits to determine the pharmacokinetics of dexamethasone phosphate and its metabolic product, dexamethasone, at low (25 microg/kg) and high (250 microg/kg) doses. Plasma samples were collected from each group of animals at different time points up to 24 h after the injection. Using a liquid chromatographic mass spectrometric method with a limit of detection of 0.5 ng/mL, the plasma concentration for dexamethasone and its prodrug compound were quantified. IV delivery showed the fastest plasma elimination, followed by SC delivery. IVT delivery exhibited a depot effect, with very low plasma levels throughout the 24-h time course. At 24 h postinjection, only the high-dose IVT and low- and high-dose SC dexamethasone injections were still detectable in the plasma.