A high pressure liquid chromatographic method for the separation and quantitative determination of sulfisoxazole in plasma was developed. Plasma samples were vortex-mixed with acetonitrile and centrifuged to obtain clear supernatant solutions. The supernate was chromatographed on a reverse phase column using acidified aqueous ethanol as mobile phase to effect separation. The separated components were detected by UV absorption at 280 nm and the peak height measurement was used for quantitative determination. This assay is simple and rapid. Each assay takes less than 8 minutes to complete and can be carried out on as little as 10 microliter plasma samples to accurately measure 2.0 microgram/ml of sulfisoxazole in plasma. This method can be used for an efficient monitoring of sulfisoxazole in plasma during the chemotherapy of bacterial infections, and also for the bioavailability study of the drug from dosage forms.