OBJECTIVE To evaluated the ocular pharmacokinetics of carnosine (CAR, a biologically active dipeptide which occurs naturally throughout the human body) 5% eye drops following topical application. METHODS CAR 5% eye drops were topically applied repeatedly (50 μL × 4) at an interval of 5 min. Aqueous humor and lens were collected after 5, 15, 30, 45, 60, 90, 120, 150, and 180 min. CAR concentration was determined by high performance liquid chromatography-tandem quadrupole mass spectrometer (HPLC-MS/MS). RESULTS CAR concentration in treated eyes was significantly higher than control eyes. Peak concentration (C(max)) of carnosine in treated aqueous humor occurred 60 min following topical administration, with the administrated concentration (total-endogenous concentration) of 40.9 ± 18.9 μg/mL. The area under the concentration-time curve between 0 and 180 min (AUC(0-180)) was 3,276.8 (μg/mL) × min. CAR concentration in treated lens rises to effective level rapidly and changes slightly with time after topical administration. The administrated concentration of car in lens at the last time point (180 min, 1.92 ± 1.65 μg/mL) was not significantly different with the highest value (15 min, 2.11 ± 1.83 μg/mL). CONCLUSIONS CAR 5% eye drops were likely to be absorbed into aqueous humor efficiently and accumulated in lens. More attention should be put onto enhancing the penetration of CAR into lens capsule.