A simple and precise reversed-phase high performance liquid chromatography (HPLC) method for the determination of cefuroxime in human plasma was developed and validated. Using cefazolin as an internal standard (IS), 0.25 ml plasma samples were deproteinized with 90 µl of 3% trichloroacetic acid in methanol, the supernatant was extracted with 150 µl acetonitrile, and 100 µl of the second supernatant were injected into the HPLC system. Separation was achieved on Atlantis dC18 column with a mobile phase composed of 0.01 M cetyltriethylammonium bromide, 0.01 M dipotassium hydrogen phosphate (pH 6.5), and acetonitrile (30:30:40, v:v:v). The mobile phase was spiked with triethylamine (10 µl/L) and delivered at 1.0 ml/minute. The eluent was monitored spectrophotometrically at 278 nm. No interference with cefuroxime and IS peaks by extracted blank plasma components or commonly used drug was observed. The relationship between cefuroxime concentration and peak height ratio of cefuroxime to the IS was linear over the range of 0.25-14.0 μg/ml. Coefficient of variation and bias were ≤ 12.6% and ≤ 11.0%, respectively. Mean extraction recovery of cefuroxime and the IS was 99% and 95%, respectively. The method was applied to assess the stability of cefuroxime under various conditions encountered in the clinical laboratory. Cefuroxime stability in processed samples stored at room temperature for 24 hours or 48 hours at -20 °C, and in unprocessed samples for 24 hours at room temperature or 14 weeks at -20 °C was ≥ 96% and ≥ 83%, respectively.