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A novel chromatographic method, ultra-performance liquid chromatography (UPLC) was improved to determine sulfamethoxazole and trimethoprim in a two-component tablet formulation. In the implemented of the method, the chromatographic parameters were optimized by using the experimental design and optimization procedure. The central composite design and fitting model was applied to identify the suitable chromatographic conditions providing a desirable elution of sulfamethoxazole and trimethoprim in a chromatogram. In the central composite design, temperature, flow rate and buffer% were selected as the effective factors on the chromatographic resolution. The buffer system was the mixture of 0.1 M CH3COOH and CH3COONa (pH 4.75) in mobile phase system. In the optimization step, the chromatographic conditions were found to be 0.20 mL/min for flow rate, 38.0 °C for the column temperature and 66% for the acetate buffer system (v/v) in the mobile phase. Analysis of the investigated drugs was accomplished on a stationary phase based on Waters BEH C18 column (50 mm-2.1 mm, 1.7 mm i.d.). In the validation step, recovery study was performed by analyzing the synthetic binary mixture of sulfamethoxazole and trimethoprim. Recovery results were found as 99.12% for sulfamethoxazole and 99.44% for trimethoprim. Assay results showed that the optimized chromatographic technique was very suitable for the quantitation of sulfamethoxazole and trimethoprim in tablets. [ABSTRACT FROM AUTHOR]