A simple and reproducible high-performance liquid chromatographic method was developed for simultaneous determination of sulfamethoxazole (SMX) and trimethoprim (TMP) in human plasma. The method entailed injection of the samples after deproteination with perchloric acid and subsequent neutralizing. Primidone was used as internal standard. Chromatography was performed on a C18 column (250 mm × 4.6 mm, 5 μm) under isocratic elution with 50 mM aqueous sodium dihydrogen phosphate–acetonitrile–triethylamine (100:25:0.5, v/v), pH 5.9. Detection was made at 240 nm and analyses were run at a flow-rate of 1.5 ml/min at a temperature of 35 °C. The recovery was 83.4, 88.5 and 98.2% for TMP, SMX and internal standard, respectively. The precision of the method was 2.6–9.8% over the concentration range of 0.125–2 μg/ml for TMP and 0.39–50 μg/ml for SMX. The limit of quantification (LOQ) in plasma was 0.125 and 0.39 μg/ml for TMP and SMX, respectively. The method was used for a bioequivalence study.