A reverse phase high performance liquid chromatographic (HPLC) assay is used to quantitate N1-acetyl sulfisoxazole and the related manufacturing impurities sulfisoxazole, N4-acetyl sulfisoxazole and N1,N4-diacetyl sulfisoxazole. The HPLC separations are achieved using a microparticulate octadecylsilane column with a ternary aqueous acetic acid: acetonitrile: methanol mobile phase. The HPLC analysis of the drug potency is specific, precise (RSD values 0.3 to 1.5%) and agrees with USP XX sodium nitrite titration assays. The HPLC assay of the related manufacturing impurities requires less than 12 min. per injection and has precision (RSD values) ranging from 1.1 to 36.2%. The manufacturing impurities are detectable to less than 0.1% using the HPLC procedure. A thin layer chromatographic (TLC) procedure is described to compliment the HPLC quantitation of the manufacturing impurities.