N1-acetyl sulfisoxazole (N1AS), a dihydropteroate synthase inhibitor is known to be biotransformed primarily to sulfisoxazole, partly to N4-acetyl sulfisoxazole (N4AS), and likely also to diacetyl sulfisoxazole (DAS) and other compounds. Although its clinical use has been limited due to urolithiasis, some countries still use the drug in combination with trimethoprim in cattle. A liquid chromatographic method using ultraviolet detection was developed for the simultaneous determination of four substances for the first time. Four analytes and sulfamethoxazole (IS) were separated on a reversed-phase column with gradient elution of a mobile phase. Because DAS and N1AS in plasma were changed very quickly into N4AS and sulfisoxazole, respectively, and esterase inhibitors (sodium fluoride and eserine) could not prevent the transformation, sulfisoxazole and N4AS were monitored in rat plasma following a single oral administration of N1AS and sulfisoxazole in five rats. The relative bioavailability of N1AS to sulfisoxazole was about two, indicating that a half-dose of N1AS would be equivalent to a dose of sulfisoxazole to achieve the same systemic exposure to sulfisoxazole.