Abstract: A method of HPLC-MS/MS was established for the determination of glyphosate and aminomethylphosphonic acid (AMPA) in soil. The sample was extracted by KOH (0.6 mol/L), purified by C18-SPE, and then pre-column derivatization by FMOC-Cl. The chromatographic separation was performed on C18 column with gradient elution using acetonitrile and ammonium acetate (5 mmol/L) as mobile phases. Mass spectrometric acquisitions were carried out by means of multiple reaction monitoring (MRM) in the electrospray negative ionization mode. The results showed that the calibration curve of glyphosate and AMPA was linear within the range of 1.6-200 μ g/L. The limits of detection (LOD, S/N=3) were 0.80 μ and 0.94 μ g/kg respectively, and the limits of quantification (LOQ, S/N=10) were 2.6 and 3.0 μ g/kg respectively. The recoveries in different samples at three spiked concentration levels ranged from 84% to 104 % with the relative standard deviations from 2.8% to 7.5%. The current method has been applied in the analysis of some soil samples. The results showed that the detection rate of glyphosate and AMPA were 38.9% and 61.1%, respectively.