AIM: To evaluate the levofloxacin eye drop into human eye penetration, levofloxacin eye drop concentrations in human ocular aqueous of 33 patients undergoing cataract surgery were measured by high performance liquid chromatography (HPLC). METHODS: Totally 33 volunteer patients who scheduled for phacoemulsification surgery received one drop of levofloxacin every 6h for 3d before and on the day of surgery, administration of drug was stopped 1h before surgery. Levofloxacin concentration in aqueous humor was measured by HPLC method with fluorescence detector. RESULTS: A simple, effective and sensitive HPLC method for determination of levofloxacin in human ocular aqueous was validated. Linearity was shown for levofloxacin concentration over a wide range of 1.95×10-3-1.50 μg/mL. The mean aqueous level of levofloxacin was 0.3399±0.03405 μg/mL. CONCLUSION: Results from the present study demonstrate that topical administration of levofloxacin 0.5% before cataract surgery with routine dose (one drop every 6h) unable to reach MIC90 for most common microorganism causing acute bacterial endophthalmitis.