Objective:A selective, reproducibility, efektive, sensitive, simple and fast of High Performance Liquid Chromatography (HPLC) was developed, optimized and validated to analyze 25-Desacetyl Rifampicin (25-DR) in human urine which is a tuberculosis patient. Materials and Methods: The separation was performed by HPLC Agilent Technologies with column Agilent Eclipse XDB-C₁₈ and mobile phase of 65:35 v/v methanol:0.01 M sodium phosphate buffer pH 5.2, at 254 nm and flow rate of 0.8 ml/min. Result: The mean retention time was 3,016 minutes. The method was linear from 2–10 μg/ml 25-DR with correlation coefficient of 0.9978. Standard deviation, relative standard deviation and coefficient variation of 2, 6, 10 μg/ml 25-DR were 0-0.0829, 0-3.1752, 0-0.0317%, respectively. The recovery of 5, 7, 9 μg/ml 25-DR was 80.8661, 91.3480 and 111.1457%, respectively. Limits of detection (LoD) and quantification (LoQ) were 0.51 and 1.7 μg/ml, respectively. Conclusion: The method has fulfilled the validity guidelines of the International Conference on Harmonization (ICH) bioanalytical method which includes parameters of specificity, linearity, precision, accuracy, LoD and LoQ. The developed method is suitable for pharmacokinetic analysis of various concentrations of 25-DR in human urine.