A simple, rapid and specific method using high performance liquid chromatography with fluorescence detector (HPLC-FLD) was developed for quantification of glucosamine in supplements after derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl). The chromatographic separation was achieved on a C18 column (150 mm × 4.6 mm × 5 µm) using a gradient mobile phase containing acetonitrile and water at a flow rate of 1.0 mL/min. The FLD detector was operated at excitation and emission wavelengths of 265 and 315 nm, respectively. The analytical method was validated for specificity, linearity, accuracy, and precision. The detector response for glucosamine was linear over the selected concentration range from 6.7 to 135 ppm with a correlation coefficient of 0.9988. The repeatability (RSD, n = 6) and reproducibility (RSD, n = 4) were from 2.54 to 3.23 % and from 2.24 to 3.07 %, respectively. The recovery was between 98.0 and 101.5 %. In addition, we also joined the interlaboratory proficiency testing program (code: H21.11) organized by the National Institute of Food Control with good result (z-score value was - 0.89 < 2). The method was successfully applied for the analysis of glucosamine in 15 supplement samples.