Abstract: An HPLC method was established for the determination of pyrazinamide and its related substances. The method was carried out on a TSK-gel ODS-gel C₁₈ column(250 mm×4. 6 mm, 5 μm)with a mobile phase consisting of methanol-water(pH 3. 0 adjusted with glacial acetic acid)(8 ∶92)at the flow rate of 1. 0 mL/min. The detection wavelength was set at 268 nm and the column temperature was kept at 35 °C. There was fair resolution between pyrazinamide and its related substance. The calibration curves were linear over the range of 0. 4 μg/mL and 250 μg/mL(r=1. 00). The limits of detection and quantitation were 6. 5 ng/mL and 40. 5 ng/mL, respectively. The contents data of nine batches of pyrazinamide determined by the developed method were consistent with those measured by non-aqueous potentiometric titration(BP 2012). The established HPLC method is specific, accurate, durable and easy to operate, has been proposed to be used in the Chinese Pharmacopoeia and the International Pharmacopoeia. It could be applied for the determination of pyrazinamide and its related substances.