A method for the analysis of glufosinate ammonium (GLUF) and its metabolite 3-methylphosphinicopropionic acid (MPPA) in human serum by gas chromatography–mass spectrometry (GC–MS) was developed. Employing a mixed-mode cartridge with both anion exchange action and weak nonpolar interaction, we extracted GLUF and MPPA from the serum and carried out GC–MS analysis of their tert-butyldimethylsilyl derivatives. The detection limits of GLUF and MPPA were 10 pg and 1 pg, respectively. Full mass spectra of 100 pg GLUF and of 10 pg MPPA were easily obtainable. The recovery rate of 90.0 ± 11.9% (or better) when the serum concentrations of GLUF and MPPA were 10–0.1 µg/mL. Results of 23 serum samples, from patients with GLUF poisoning, measured by this method correlate well with those derived from the conventional high-performance liquid chromatography method (r = 0.996). The developed GC–MS method is likely to become a useful analytical technique in clinical settings.