This study describes a method for the sensitive and selective determination of glyphosate and aminomethylphosphonic acid (AMPA) in water samples. The method involves a derivatization step with 9-fluorenylmethylchloroformate (FMOC) in borate buffer and a detection based on liquid chromatography coupled to electrospray tandem mass spectrometry (HPLCESI-MS/MS). A volume of water samples was derivatized and then the excess of FMOC-Cl was eliminated with an extraction with ethyl acetate. The derivatized analytes were preconcentrated by solid phase extraction prior to the HPLC-ESI-MS/MS analysis.