A simple HPLC assay was evaluated for analysis of ofloxacin in biological fluids. Mobile phase contained methanol (40%) and phosphate buffer (pH 3.5, 60 mmol/1) and a common C18column was used. Detection was set at 280 nm (UV). Biological fluids (50 μl) were prepared for assay by protein precipitation with acetonitrile (1/1, vol/vol). The assay is linear from 2000 to 10 ng/ml and sensitive to 10 ng/ml. The mean recovery of ofloxacin from serum was 99.4%. The coefficient of variation was < 4.0% for same-day precision and <6.8% for assay-to-assay precision. Because the assay requires only small specimen volumes and minimal sample preparation, this assay provides a simple to use, sensitive and specific method for quantification of ofloxacin in biological fluids. © 2011 by Walter de Gruyter GmbH & Co. All rights reserved.