A simple and reliable thin-layer chromatographic method for the determination of sulfamethoxazole, trimethoprim, and impurities of sulfanilamide and sulfanilic acid is developed and validated. The effect of the polarity and the composition of eluents on the separation of this mixture is investigated. A chloroformn-heptane-ethanol (3:3:3, v/v/v) solvent system is used for a quantitative evaluation of chromatograms. The chromatographic zones, corresponding to the spots of sulfamethoxazole, trimethoprim, sulfanilamide, and sulfanilic acid on the silica gel plates, are scanned in the reflectance/absorbance mode at 260 nm. The method is found to be reproducible and convenient for the quantitative analysis and purity control of co-trimoxazole in its raw form and in its dosage forms. quantitative assay of the active drugs sulfamethoxazole and trimethoprim in pharmaceutical dosage forms. There are no reports of the simultaneous TLC assay of co-tri moxazole and impurities of sulfanilamide or sulfanilic acid in the literature. The major impurities in sulfamethoxazole drug substances and dosage forms are sulfanilamide and sulfanilic acid. The current United States Pharmacopeia 23rd revision (USP XXIII) specifications allow 0.2% (w/w) sulfanilamide and sulfanilic acid in sulfamethoxazole drug substances and 0.5% or 0.3% sulfanilamide and sulfanilic acid in dosage forms, re spectively (13). Therefore, we describe a simple, accurate, and quick TLC technique for the simultaneous determination of co-trimoxa zole and its impurities in sulfamethoxazole drug substances and in its dosage forms.