AbstractMetformin (MtF) is a treatment used for type 2 diabetes. Lactic acidosis (LA) is a frequent complication that can be either induced by or associated with elevated MtF plasma concentrations. When coupled with a mass spectrometry (MS) system, the probe electrospray ionization (PESI) method allows direct and rapid analysis of different types of matrices without pretreatment. In this study, we developed a PESI–MS method for the determination of MtF in plasma. We used a tandem mass spectrometer equipped with a PESI source in the reaction monitoring mode for the quantitation of MtF. MtF-d6 was chosen as the internal standard (IS), following an isotope dilution (ID) approach. The method was fully validated with six concentration levels (0.5–50 mg/L). The matrix effect was evaluated for each level, and the specificity was tested with a mix of potential co-medications. Using patient samples, the performance was compared with two classical LC–MS-MS and LC–diode array detector (DAD) methods used in external labs. Sample preparation consisted in mixing 10 µL plasma in 1,000 µL ethanol/ammonium formate buffer including MtF-d6 at a fixed concentration of 5 mg/L. The total run time was 0.31 min. ID gave satisfactory results of accuracy and precision (min–max: −12.1 to 15.8% and 1.0–17.1%, respectively). The matrix effect was fully corrected by the internal standard (bias < 1%). The specificity study also reported satisfactory results. Finally, in a representative group of 29 patients (55% with a concentration <5 mg/L, 38% with a concentration >5 mg/L and 7% not detected), we observed almost identical results when comparing LC–DAD and LC–MS-MS to PESI–MS (r2 > 0.99). We propose a specific, sensitive, accurate and ultrafast solution for the measurement of MtF in patient plasma, with no sample preparation or calibration curve building. This could be helpful in a core lab when rapid diagnosis of LA is needed.