Objective: A simple, precise and accurate UV-spectrophotometric method is developed and statistically validated for estimation of Testosterone in gel formulation. The proposed method includes using regression equation, area under curve (AUC), first order derivative and second order derivative spectroscopic method.Methods: based on measurement of absorbance at a selected wavelength using UV-visible spectrophotometer with 1cm matched quartz cell and acetonitrile as a solvent. All developed methods obeyed Beer's-lambert's law in the concentration range of 5-25μg/mL, with correlation coefficient value less than 1. Results:The percent amount of drug estimated was nearly 100%, found to be a good agreement with label claim of marketed gel formulation. The recovery study was carried out at three different levels, the validation study data was found to be statistically significant as all the statistical parameters are within the acceptance range (% RSD <2.0 and S.D. <±2.0). Conclusions:The results of estimation and validation parameters like accuracy, precision, ruggedness, linearity and range were studied for all the developed methods and were found to be within limits. The results obtained were statistically compared using paired t-test and one way ANOVA analysis. The proposed method can be adopted for routine quality control for estimation of drug in formulation.