Purpose: This study evaluated intraocular penetration of fluoroquinolones (FQs) viz, ofloxacin, pefloxacin, lomefloxacin and sparfloxacin in aqueous, vitreous humors, and plasma after oral/intravenous administration in the subjects undergoing vitrectomy. Methods: Ofloxacin, pefloxacin, lomefloxacin and sparfloxacin were included for oral administration. Only ofloxacin and pefloxacin included for intravenous study. Each group was sub-divided into 3 time points (3, 6 and 12hrs post dose). For each time point, 8 eyes (8 patients) were included and a total of 144 patients were enrolled. Intra-operatively, 80-110μl aqueous, 100-250μl vitreous and 2ml of venous blood samples were collected. Plasma was separated and all samples were stored at -70°C and subjected for quantification using HPLC and compared with their known MIC90 of common ocular pathogens. Results: In oral 400mg single dose, among all 4 FQs, ofloxacin showed a maximum concentration at 3 hours in aqueous [0.98(0.12)μg/ml] and vitreous [0.78(0.12)μg/ml]. Intravenous infusion of 400mg ofloxacin also showed the similar levels in the humors of the eye. Despite low plasma levels, lomefloxacin showed higher aqueous to plasma (a/p) and vitreous to plasma (v/p) ratio. Conclusion: FQs 400mg (as iv and oral) as a single dose could not reach the desired therapeutic concentration (above MIC90 for all ocular pathogens) in aqueous and vitreous humors. The concentration reached in the humors may not be directly dependent on their plasma levels. Relatively non-polar FQs such as lomefloxacin and sparfloxacin showed better a/p and v/p ratios as compared to their polar analogs such as pefloxacin and ofloxacin.