Pioglitazone hydrochloride and Glimepiride is antidiabetic drug. A sensitive, precise, accurate and simple first order zero crossing UV spectrophotometric method has been developed for simultaneous Estimation of Pioglitazone hydrochloride and Glimepiride in bilayer tablet dosage form. The quantification was achieved by the first-order derivative spectroscopy method at 225 nm (zero cross point of Glimepiride) for pioglitazone and 248 nm (zero cross point of Pioglitazone) for Glimepiride. Pioglitazone HCl (R2=0.9912) and Glimepiride (R2=0.9964) shows Linearity in a concentration range of 5-30μg /ml and 4-20μg/ml respectively. Procedure does not require prior separation of layers of tablet formulation. LOD values for Pioglitazone HCl and Glimepiride are found to be 0.0187μg/mL and 0.132μg/mL, respectively. LOQ values for Pioglitazone HCl and Glimepiride are found to be 0.056μg/mL and 0.40 μg/mL, respectively. The results of analysis have been validated statistically and recovery studies carried out in the range 80-120% to confirm the accuracy of the proposed method. The relative standard deviation was found to be <2.0%. The Proposed method is recommended for routine analysis since it is rapid, simple, accurate and also sensitive and specific.