The object of this work was to optimize and validate an analytical method for the analysis of glyphosate and its main metabolite AMPA (aminomethylphosphonic acid) in natural and drinking water, for sanitary control. The method uses a derivatization step which transforms glyphosate and AMPA into fluorescent products by reaction with 9-fluorenylmethylchloroformate (FMOC-CI). The kinetics and yield of this reaction were studied by use of different samples. The derivatization products were injected directly into the liquid chromatograph and separated on an amino-functionalized silica gel column. The performance of the method (detection limit, quantification limit, linearity, and relative standard deviation) was studied and the results obtained showed the method was suitable for routine analysis of glyphosate and AMPA. An interlaboratory test with five laboratories confirmed that good results were obtained by use of this method.