Abstract: OBJECTIVE To compare determination of glucosamine using USP method, HPLC derivitization method and UV method, and inspect their actuality and reliability. METHODS ①Refer to USP28,C8 column(250 mm×4.6 mm, 5 μm) was used with acetonitrile-phosphate buffer(pH 3.0)(40∶60) as mobile phase, detect wavelength was 195 nm and column temperature was 30 ℃. ②HPLC derivitization method used C18 column(250 mm×4.6 mm, 5 μm), the mobile phase consisted of acetonitrile-phosphate buffer(pH 2.5)(40∶60), the detected wavelength of 265 nm, and the temperature of 30 ℃. Glucosamine was derivatized with 9-fluorenylmethoxycarbonyl succinimide (FMOC-Osu) solution and the derivative was detected under the chromatography conditions. ③In the UV method, potassium ferricyanide reacted with glucosamine in sodium carbonate solution and determine the absorption value at 420 nm. RESULTS The linearity of three methods was good. The precision and recovery of HPLC derivitization method and UV method were better than USP method. The result of USP determination of glucosamine sulfate and its preparation was lower. CONCLUSION The result of USP method is not perfect. HPLC derivitization method and UV method are applied to determine the content of drug substances of glucosamine, and the first is more suitable to determination of glucosamine preparation.