A combination of Melatonin and Pyridoxine is in called as Anti-convulsant agent. The present work deals with the RP-HPLC methods for simultaneous determination of Melatonin and Pyridoxine in pharmaceutical formation. Attempts were made to develop RP-HPLC method for simultaneous estimation of Melatonin and Pyridoxine from its formulation. For the RP HPLC method, Agilent with auto sampler Gradient System DAD Detector and C18 column with 250mm x4.6 mm i.d and 5μm particle size Acetonitrile 40%:60% water with 0.1% TEA (PH 6.2 with OPA) was used as the mobile phase for the method. The detection wavelength was 265 nm and flow rate was 1.3.3 ml/min. In the developed method, the retention time of Melatonin and Pyridoxine were found to be 2.008 min and 3.042 min. The developed method was validated according to the ICH guidelines. The linearity, precision, range, robustness was within the limits as specified by the ICH guidelines. Hence the method was found to be simple, accurate, precise, economic and reproducible. Keyword: Melatonin, Pyridoxine, RP-HPLC.