A fast LC-MS/MS application for the determination of glyphosate and its metabolite amino-methyl-phosphonic acid (AMPA) with direct injection and without derivatization is presented. The separation and selective determination of AMPA and glyphosate was carried out on a specifically dedicated chromatographic method from DASP-AB Sciex. A sensitive and selective detection was ensured by multiple reaction monitoring (MRM) acquisition of the product ions 63 (m/z) and 79 from AMPA precursor ion 110 and product ions 63 and 81 from glyphosate precursor ion 168. Isotope-enriched molecules were used as internal standard, monitoring the product ion 63 from both precursor ions 112 and 171 for ¹³C- and ¹⁵N-enriched AMPA and glyphosate, respectively. Sensitivities down to 0.02 μg/l were obtained with repeatability within 6% in the investigated interval of 0.02-0.05 μg/l. The analytical recoveries determined at 0.02 and 0.05 μg/l were in the 80-120% range.