Introduction: A UV-spectrophotometric method has been developed for the quantitative estimation of fluconazole. The present study describes development and validation of UV-spectroscopic method for estimation of fluconazole reference substance and validation of detection method. Method: During development of analytical method HCl: water (6 different concentrations). The detection length (cmax) was found to be 260nm. Calibration curves were prepared. The proposed method obeys Beer’s law in the range of 5-50mkg/ml. Absorption maxima was determined with 10 mkg/ml by scanning in the range of 200-400nm. Results: Percent recovery studies are in the range of 95,12%-101.89%. The method was validated in terms of specificity, linearity, accuracy, range, precision, repeatability, robustness, stability of analytical solution. Results of analysis were validated statistically and by specificity studies. From that it was observed that there is no interference of blank, excipients during the estimation of reference substance in formulation. Conclusion: This shows the adaptability of the method for routine estimation of fluconazole reference. Keywords: Fluconazole, method development, validation, spectrophotometry