Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis. Nicotinic acid derivatives such as isoniazid have the strongest anti-tuberculosis properties. For pharmacokinetics studying of isoniazid (INH), a method is needed to determine the levels of INH in plasma. The aim of this research is to partial validate of high performance liquid chromatography (HPLC) for analysis of INH in rat plasma.For the preliminary study, rat plasma was used. The HPLC system used is a stationary phase C18 with length 250 mm and temperature of 30 °C, mobile phase hexane sulphonate acid 20mM pH 2.47–methanol (65:35). The analytical parameters in partial validated were linearity, lower limit of quantifi cation (LLOQ), precision, accuracy, and recovery. The results of linearity test of INH showed r value of 0.9996. LLOQ of this methodwas 0.1258 μg/mL.The resulting accuracy and precision value met FDA requirements with a percent recovery ranging from 96.57–107.99%. The HPLC system was a valid method for analysis of INH in rat plasma.