Modern agricultural technology is based on a package that combines direct seeding and glyphosate with transgenic crops (soybean, maize, and cotton). Because of widespread glyphosate use, it is important to assess its impact on the environment. However, glyphosate's unique physicochemical characteristics make determination difficult at residue concentrations, especially in soils with high organic matter content, such as the soils from the central eastern region of Argentina, where strong analytical interferences are normally observed. The aim of this work was to compare the efficiency of two extraction methods of glyphosate using representative soils from Argentina. One method is based on the use of a phosphate buffer as the extracting solution and dichloromethane to minimize matrix organic content. The other method uses potassium hydroxide for the soil extraction of analytes and involves a cleanup step using solid phase extraction to minimize analytical interferences. Both methodologies involve a derivatization with 9-fluorenyl-methyl-chloroformate in borate buffer, the use of isotope labeled glyphosate as an internal standard, and detection based on ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry. Recoveries obtained for soil samples spiked at 100 and 1000 μg kg−1 were satisfactory in both methods (70%–120%). However, significant differences were observed in the effects on the organic matrix. The solid phase extraction cleanup step was insufficient to remove the interferences, whereas the dilution and the cleanup with dichloromethane were more effective in minimizing the ionic suppression.