The HPLC method was developed and validated for simultaneous estimation of Rifampicin and Isoniazid. The mobile phase consisted of pH 3.5 phosphate: Methanol: water (45:30:25). The linearity range of Rifampicin was found to be 9-45 μg/ml and Isoniazid 6-30 μg/ml. The calibration curve was plotted, and regression equation of Rifampicin was found to be y = 78,303.72x - 48,524.90 with correlation coefficient (r2) of 0.9992 and Isoniazid y = 47,353.45x + 51,031.70 with correlation coefficient (r2) of 0.9993. Detection was done at 239 nm and the retention time of Rifampicin was found to be 2.8 min and Isoniazid 3.7 min with the flow rate of 0.8 ml/min. From accuracy study % recovery of Rifampicin was found in the range of 98.72-100.98% and Isoniazid is 98.77-101.45% which is in the limits according to the ICH guidelines. The limit of detection and limit of Quantitation of Rifampicin is 0.24µg/ml – 0.72µg/ml and Isoniazid is 0.06 µg/ml – 0.19 µg/ml respectively. The method was found to be simple, linear, rapid, accurate, precise, reproducible and robust. The % RSD was found within the limit as per ICH guidelines. All the analyzed validation parameters showed acceptable data with satisfactory correlation co-efficient and lower % RSD as per the ICH guidelines. The developed method can be utilized by industry for quantitative simultaneous estimation of Rifampicin and Isoniazid as bulk and in tablet dosage form.