A sensitive and selective reversed-phase HPLC method for analysis of ketorolac in aqueous and vitreous humor from the human eye has been developed and validated. Chromatographic separation was achieved on a 250 mm × 4.6 mm i.d., 5-μm particle, C₁₈ analytical column. Photo diode-array detection was performed at 314 nm. Response was a linear function of ketorolac concentration from 10 to 800 ng mL⁻¹. The limits of detection (LOD) and quantification (LOQ) were 3.0 and 10 ng mL⁻¹, respectively. Intra-day and inter-day bias were less than 2.05 and 2.28%, respectively, and intra-day and inter-day RSD were no higher than 3.60 and 5.80%, respectively. Fluid obtained from patients eyes’ after topical application of Acular eye drops before retina decolman surgery was analyzed by use of the method. The method enabled successful quantification of levels of ketorolac in aqueous and vitreous humor.