Isoniazid (INH) is metabolized by N-acetyl transferase-2 into acetyl isoniazid (Ac-INH). The acetylation rate is different in each ethnic, therefore it is very interesting to study of INH acetylation. The aims of this study were to synthesize Ac-INH and the produced Ac-INH was used as a working standard for the pilot study of INH acetylation in Melanesian healthy subjects from Indonesia. Methods: Ac-INH can be simply synthesized by acetylation reactions between INH and acetic anhydride. The synthesis results were characterized by organoleptic observation, purity test, UV-Vis spectrophotometry analysis, FTIR analysis, and mass spectrometry analysis. The pilot study of INH acetylation used high performance liquid chromatography to analyze the levels of INH and Ac-INH in blood of Melanesian healthy subjects. The acetylation rate of INH was determined by the ratio between Ac-INH and INH concentrations at 3 h after administration of INH. Results: The acetylation reaction to synthesize Ac-INH has been performed at temperature of 60°C for 30 min. The obtained Ac-INH had the same quality specifications with reference standard of Ac-INH. The average of ratio between of Ac-INH and INH concentrations was