A simple, rapid, selective and specific high-performance liquid chromatography (HPLC) method with fluorescence detection was developed for determination of glucosamine sulfate in human plasma and application to a bioequivalence in healthy volunteers. Precipitation of plasma was accomplished with acetonitrile to separate interfering endogenous products from the compound of interest. After vortex mixing and centrifugation, the supernatant was transferred and derivatized with 9-fluorenylmethoxycarbonyl chloride-acetonitrile solution in borate buffer (pH=8.0) at 30 degrees C for 30 min. The chromatographic separation was performed on a Diamonsil C18 column (150.0 mmx4.6 mm, 5 microm) with a mobile phase gradient consisting of water and acetonitrile at a flow rate of 1 mL/min. The method was linear in the range of 0.1-10.0 microg/mL with a correlation coefficient (r) of 0.9996. The limit of detection was 15 ng/mL. Inter- and intra-day precisions were <or=6.28 and 7.41%, respectively, and the accuracy ranged from 95.20 to 104.92%. Extraction recoveries of glucosamine sulfate from plasma were more than 90.4%. Plasma samples containing glucosamine sulfate were stable for 40 days at -20 degrees C and for 24 h after derivatization at 4 degrees C. The method was successfully applied to the bioequivalence study of glucosamine sulfate in healthy volunteers.