A derivatization method for the determination of glyphosate was developed by using o nitrobenzenesulfonyl chloride as derivatization reagent.The optimized conditions of derivatization were as follows:reaction temperature:25 ℃，reaction time:10 min，buffer solution:0.25 mol/L borate buffer（pH 9.0），the molar ratio of glyphosate to o-nitrobenzenesulfonyl:1∶5.The optimal chromatographic conditions were as follows：Lichrospher C18 column（5 μm，4.6 mm×250 mm），detection wavelength:230 nm，flow rate:1.0 mL/min，mobile phase:methanol-pH 5.5 0.05 mol·L-1phosphate buffer(20∶80)．Under the optimal conditions，the calibration curve for glyphosate was linear in the range of 0.05-1.0 g·L-1 with a correlation coefficient （r2）of 0.999 3.The limit of detection was 6.25 mg·L-1.The method was simple，rapid and reliable，and was suitable for the determination of glyphosate in general laboratory.