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Aim: To assess the intraocular penetration of 0.5% moxifloxacin hydrochloride into aqueous humour after oral and topical administration., Methods: A prospective, interventional study of 42 patients scheduled to undergo cataract surgery was carried out. Out of the 42 subjects, 21 were randomly categorised into Group I and received one drop of 0.5% topical moxifloxacin four times, at 15 min intervals starting 75 min before the surgery. Another 21 subjects were categorised into Group II and all subjects in this group were administered a single tablet of 400 mg of moxifloxacin, 12 h before the surgery. Estimation of moxifloxacin in aqueous samples was carried out using high-performance liquid chromatography. Results were analysed using Student unpaired 't' test and analysis of variance. The value of p<0.05 was considered to be significant., Results: Mean aqueous concentration of moxifloxacin attained in the oral group (n=21) was 0.504±0.30 μg/mL while that in the topical group (n=21) was 2.04±0.72 μg/mL, and this difference in levels was statistically significant (p<0.005). The levels attained by both the groups well exceeded the MIC90 (minimum inhibitory concentration of antibiotic required to inhibit growth of 90% of bacteria strains) levels for most of the organisms causing endophthalmitis. Penetration of moxifloxacin in aqueous in both the groups was not affected by gender, intraocular pressure or comorbidities significantly. However, aqueous levels were found to be higher among the younger subjects within the topical group., Conclusions: Moxifloxacin has an impressive spectrum of coverage and this pharmacokinetic study reinforces its potential as a prophylactic drug against intraocular infections, given the high aqueous levels post topical administration., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)