A simple and reproducible high-performance liquid chromatographic method was developed for simultaneous determination of sulfamethoxazole (SMX) and trimethoprim (TMP), which are also known as cotrimoxazole, in tablet and human plasma in vitro. The Concentration of TMP and SMX used for this study were 1:5 according to their combination as cotrimoxazole. Chromatography was performed on a C18 column (250 mm × 4.6 mm, 5 μm) under isocratic elution with acetonitrile-water-triethylamine (20:80:0.1 v/v), pH 5.9 ± 0.1 arranged by 0.2 N NaOH or 1% acetic acid. Detection was made at 240 nm and analyses were run at a flow-rate of 1.0 ml/min at a room temperature. Sulfadimidine was used as internal standard. In tablet validation, the calibration curve was linear by r values 0.9994 and 0.9996, precision by coefficient of variation (CV) were 0.85% and 0.98% also accurate by % recovery for 3 concentrations were 98% - 102% for TMP and SMX, respectively. Plasma extraction was done by deproteination with acetonitrile, mix with vortex for 40s, then centrifuge it on 12500 rpm for 15 minutes. In plasma validation, the recovery was 94.95% and 86.87% for TMP and SMX, respectively. The lower limit of quantification (LLOQ) in plasma was 150 ng/mL and 750 ng/mL for TMP and SMX, respectively. The method also fulfill the criteria for accuracy and precision intra and inter day by % diff values not exceed ± 20% for LLOQ and ± 15% for concentrations except LLOQ. On the stability study, cotrimoxazole in plasma is pronounced to be stable for 30 days.