Simple, precise, accurate, rapid and economical UV-Visible spectrophotometric analytical method for simultaneous estimation of Isoniazid and Pyridoxine has been developed. For development of the method distilled water used as solvent. The method shows maximum absorbance at 262 nm and 291 nm for Isoniazid and Pyridoxine respectively. The proposed method was validated as per ICH guideline. The linearity was established over the concentration range of 3 18 ?g/mL and 0.4 2.4 ?g/mL for Isoniazid and Pyridoxine with squared correlation coefficients (r2) 0.999 and 0.999 respectively. The percentage recovery of Isoniazid and Pyridoxine were found to be 99.92 ± 0.053 and 99.93 ± 0.134 respectively. The relative standard deviation for six replicates was found less than 2.0%. The Statistical analysis gives evidences that proposed method is suitable for routine analysis of Isoniazid and Pyridoxine in pharmaceutical formulation without any interference from the excipients.