A simple and precise reversed-phase high performance liquid chromatography (HPLC) assay for the determination of rifampicin (RIF) in human plasma was developed and validated. Using hydrochlorothiazide (HCT) as an internal standard, separation was achieved on Atlantis dC18 column with a mobile phase composed of 0.01 M monobasic sodium phosphate and acetonitrile (60:40, v:v) and delivered at a flow rate of 1 ml/minute. RIF and HCT were extracted from plasma using methyl tert-butyl ether and dichloromethane (70:30%, v:v) and monitored using photodiode array detector set at 337 nm. Relationship between RIF concentration and peak height ratio of RIF to HCT was linear over the range of 0.3-25 μg/ml. Coefficient of variation and bias were ≤9.7% and ≤6.0%, respectively. Mean extraction recovery of RIF and HCT was 95% and 90%, respectively. Stability of RIF in extracted (24 hours at room temperature or 48 hours at -20 °C) and unprocessed (24 hours at room temperature, 8 weeks at -20 °C, or 3 freeze-thaw cycles) samples was ≥91%.