Dexamethasone sodium phosphate (DSP) is increasingly used in the treatment of ocular inflammatory diseases by systemic, periocular, and recently, intravitreal injection. We have developed a method for the determination of vitreous levels of DSP by reverse phase HPLC. In this method, co-elution of vitreous proteins with DSP is resolved by a prior sample clean-up procedure using Waters Sep-Pak C18 cartridge; the protein is separated and eluted with water while DSP, paraben and prednisone are eluted with methanol. DSP in the resulting sample is then separated by reverse phase HPLC and quantified by UV absorption at 254 nm. The recovery of DSP through the sample clean-up is 68.9 ± 3.0%. DSP quantitation is linear from 0.1 mg to 1.0 mg per 1.0 ml vitreous. This method provides a simple, sensitive and reliable technique for determining the vitreous levels of DSP.