Abstract: A method of SPE-HPLC coupled with pre-column derivatization was proposed for the determination of glyphosate and aminomethylphosphonic acid in drinking water. Water sample was derived with FMOC-Cl and purified by C18 solid phase extraction column. The derivatives were separated on an Atlantis T3 column with a mixture of methanol and 0.02 mol·L-1 of ammonium acetate (60+40) as mobile phase. The wavelengths of fluorescence excitation and emission used were 266 nm and 315 nm respectively. The linear relationship between the peak area and the mass concentration of glyphosate and aminomethylphosphonic acid were in the range of 5.00-500 μg·L-1, with detection limits (3S/N) of 0.002 μg·L-1. The recovery rates measured by adding standard samples to water sample were in the range of 84.4%-92.0% and 87.8%-96.8% for glyphosate and aminomethylphosphonic acid respectively, with RSD (n=5) in the range of 1.2%-4.3% and 1.1%-4.0% respectively. The method was used for simultaneous determination of glyphosate and aminomethylphosphonic acid in water samples of water supply plants.