The potential of micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) detection for the separation and determination of phosphorus-containing amino acid-herbicides (glufosinate and glyphosate), and aminomethylphosphonic acid (the major metabolite of glyphosate), involving derivatization with fluorescein isothiocyanate (FITC) isomer I, was investigated. Different variables that affect derivatization (pH, FITC concentration, time and temperature) and separation (pH and concentration of the buffer, kind and concentration of surfactants and applied voltage) were studied. The analysis was conducted within about 8 min and the use of the nonionic surfactant Triton X-100 improved the selectivity, thus indirectly enhancing sensitivity by shifting of the interfering peaks of the FITC excess. Dynamic ranges of 2.0-3000 mug/L, limits of detection at microgram or submicrogram-per-liter level, and relative standard deviations from 4.7 to 6.4% were obtained. The ensuing method - non ionic surfactant MEKC - is a useful choice for the determination of these herbicides as it provides limits of detection similar or lower than those reported by existing chromatographic alternatives without the use of an additional preconcentration technique such as solid-phase extraction. The separation of a mixture of nine FITC-derivatized amino acids, selected as target compounds, was also carried out to assess the discrimination power of the nonionic surfactant MEKC method for the analysis of closely related anionic analytes.