The extensive use of herbicides, such as glyphosate and glufosinate, in crop production during recent decades has raised concerns about human exposure. Nevertheless, analysis of trace levels of these herbicides in human biospecimens has been challenging. Here, we describe a method for the determination of urinary glyphosate, its degradation product aminomethylphosphonic acid (AMPA), and glufosinate using liquid chromatography-tandem mass spectrometry (LC−MS/MS). The method was optimized using isotopically labelled internal standards (13C2, 15N-glyphosate, 13C, 15N, D2-AMPA, and D3-glufosinate) and solid-phase extraction (SPE) with cation-exchange and anion-exchange cartridges. The method provides excellent chromatographic retention, resolution and peak shape of target analytes without the need for strong acidic mobile phases and derivatization steps. The instrument linearity was in the range of 0.1−100 ng/mL, with R > 0.99 in the matrix for all analytes. The method detection limits (MDLs) and the method quantification limits (MQLs) were in the ranges of 0.12 (AMPA and glufosinate)−0.14 (glyphosate) ng/mL and 0.40 (AMPA)−0.48 (glyphosate) ng/mL, respectively. The recoveries of analytes spiked into urine matrix ranged from 79.1% to 119%, with coefficients of variation (CVs) of 4−10%. Repeated analysis of samples for over 2 weeks showed intra-day and inter-day analytical variations of 3.13−10.8% and 5.93−12.9%, respectively. The matrix effects for glyphosate, AMPA, and glufosinate spiked into urine matrix averaged −14.4%, 13.2%, and 22.2%, respectively. The method was further validated through the analysis of external quality assurance proficiency test (PT) urine samples. The method offers optimal sensitivity, accuracy, and precision for the urine-based assessment of human exposure to glyphosate, AMPA, and glufosinate.