A rapid screening method for simultaneous determination of phosphorus-containing amino acid type herbicides such as glyphosate (GLYP), glufosinate (GLUF), bialaphos (BIAL) and their major metabolites, AMPA and MPPA in human samples using N -Acetyl, O -Methyl derivatives with high-performance liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) was developed. Serum sample with deporoteinization with 10% trifluoroacetic acid and urine sample without pretreatment was evaporated, and then the drid analytes were directly derivatized by trimethyl orthoacetate and acetic acid at 100°C for 30 minutes. LC analyses were perfomed on a reverse phase column (Symmetry C8) in the gradient solvent system of 5 mM ammonium formate and acetonitrile.   Calibration curves of GLYP, GLUF, BIAL, AMPA and MPPA showed good linearity in the range of 0.1-5, 0.1-5, 0.2-5, 0.2-5 and 0.1-5 μg/ml in serum, and 0.1-5, 0.1-5, 0.5-5, 0.5-5 and 0.1-5 μg/ml in urine, respectively. The detection limits of GLYP, GLUF, BIAL, AMPA and MPPA in the selected ion monitoring (SIM) mode were 0.05, 0.03, 0.1, 0.2 and 0.05 μg/ml in serum, and were 0.05, 0.05, 0.4, 0.2 and 0.05 μg/ml in urine, respectively.