Abtract. Objective : The study aimed to evaluate the influence of mutation gene rpoB of Mycobacterium (M.) tuberculosis cluster I (507-534) on the elimination of 25-Desacetyl Rifampicin (25-DR) in urine of tuberculosis (TB) subjects. Material and Method : Early morning sputum was taken from patient TB before treatment. Urine was collected after 2 hours at days 7^th treatment. All sputum were sequensing at Macrogen Korea Laboratory. Urine were analyzed by High Performance Liquid Chromatography (HPLC) using method of Lily et al. Result :  Mean (standard deviation) for mutation and non mutation of rpoB M. tuberculosis group were 7.6 (4.4) and 4.6 (1.7), respestively. Shapiro-Wilk test showed normal distributed data, with p value 0.3. Independent t test performed significancy 0.167 and confidence interval (CI) from -1.6 to 7.7. Conclusion : The mutation of rpoB M. tuberculosis in this study did not affect urine metabolite of 25-DR statistically and clinically.