A NP-HPLC is a simple, accurate, and rapid with reliable UV/PDA detector for the simultaneous estimation and validation of calcitriol showed the maximum absorbance at 265 nm. The separation was done by using the column zodiac silica, 4.6 × 250 mm, 5µm. The mobile phase used consisted of Mix 1 volume of propan-1-ol, 2 volumes of methanol, 40 volumes of n-hexane and 60 volumes of ethyl acetate).The chromatographic condition were performed at the flow rate of 1.2ml/min by isocratic elution mode. According to ICH guidelines, the method is validated for accuracy, linearity, precision, intermediate precision, robustness, specificity system suitability, forced degradation study, effect of variation in flow rate, stability of analytical solution and filter validation .The calibration curve were linear at the concentration of 50–150µg/ml for calcitriol were 0.9978 respectively. The percentage recovery of calcitriol were found to be 99.68% to 101.08 % respectively. The developed method was applied successfully for the HPLC analysis of simultaneous estimation and validation of calcitriol combined dosage form.