A simple, accurate, precise and rapid UV spectrophotometric simultaneous equation method has been developed for the simultaneous estimation of Melatonin and Pyridoxine in tablet dosage form. The stock solution was prepared in methanol. 243 and 271nm, an absorbance maxima were selected for Melatonin and Pyridoxine respectively. Beer’s law obeyed the concentration range of 3-9 μg/ ml and 10-30 μg/ml, for Melatonin and Pyridoxine. The results of analysis were validated statistically and by recovery studies. Accuracy and precision are within the limit. The % RSD for the recovery study was less than 2. The developed method was validated as per International conference Harmonization (ICH) guideline for its accuracy, precision, Limit of detection and Limit of quantitation. The proposed method can be effectively applied for the simultaneous estimation of both drugs in tablet dosage form.