A method was developed to determine glyphosate, aminomethylphosphonic acid (AMPA), and glufosinate in soil. The worldwide use of this herbicide in agricultural activities, and its known negative effects on both the environment and health, have generated interest in the establishment of methodologies for its determination in several matrices at trace level. The development of analytical methods for the determination of glyphosate, AMPA, and glufosinate is challenging due to its present amphoteric properties, high solubility in water, low molecular weight, high affinity to the ions presents in the soil, and lack of chromophore groups in its structure, making its quantification difficult. The proposed method exhibits a linear range from 5.0 to 600 µg/kg with limits of detection of 1.37, 0.69 and 1.22 μg/kg, limits of quantification of 4.11, 2.08, and 3.66 μg/kg for glyphosate, AMPA, and glufosinate, respectively, and adequate repeatability and reproducibility (coefficients of variation <8.0% and recovery percentages between 93.56% and 99.10%). The matrix effect was calculated for each analyte, proving to be a good alternative for the determination of these contaminants. The described method was applied to 46 soil samples collected from crop fields in Hidalgo, Mexico, with concentrations varying from not detected to 4.358 μg/kg (for AMPA).