Glufosinate and glyphosate, which are non-selective herbicides that include an amino acid moiety in their structures, are frequently used worldwide to control unwanted vegetation. Unfortunately, these readily available herbicides are also used by people to commit suicide, and thus represent important chemicals of interest in the fields of clinical medicine and forensics. Because of the high water solubility of these herbicides, most analytical methods for their detection require a derivatization step, which results in longer analysis times. Therefore, derivatization-based methods do not currently contribute to judgements on treatment decisions in emergency medicine. In this study, we addressed this limiting factor by developing an ultra-rapid and simple analytical technique using a combination of probe electrospray ionization (PESI) and tandem mass spectrometry (MS/MS), which gives quantitative results within 0.3 min. Herbicide standards were added to human serum that was then subjected to analysis (N = 5 per concentration). The analysis was repeated daily over eight consecutive days. The limit of detection (LOD) was 0.59 μg/mL for glufosinate and 0.20 μg/mL for glyphosate. The limit of quantitation (LOQ), i.e., the lowest point on the calibration curves, was 1.56 μg/mL for both the herbicides. The matrix effects were observed at three different concentrations (between 95.7%-104% for glufosinate, and between 90.7%-95.7% for glyphosate). When applied to samples taken from actual poisoning cases (six samples for each herbicide), the present method gave almost the same quantitative values as those obtained by conventional high-performance liquid chromatography with fluorescence detection. Thus, we believe that PESI-MS/MS could emerge as a rapid diagnosis method in the clinical emergency field.