We developed and validated an assay for determination of glyphosate (GLYP) and glufosinate (GLUF) in human serum. Serum samples were extracted by using a MonoSpin® TiO column and analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). MonoSpin® TiO tends to specifically bind to phosphate groups. The assay was linear over a concentration range of 1–250 μg/mL. The recoveries for the 2 compounds were 1.6%–2.3%. The intra- and inter-day variations were <15%. Precision and accuracy were 5.6%–12.7% and 97.0%–103.9%, respectively. The validated method was applied to quantify the GLYP and GLUF content in the serum of GLYP and GLUF-poisoned patients. In conclusion, the method was successfully applied for accurate determination of GLYP and GLUF in serum obtained from patients with GLYP and GLUF poisoning.