A simple, fast and sensitive quantification method for the drug metformin in dog serum was developed using flow injection analysis (FIA)-tandem mass spectrometry (MS/MS). The method was fully validated according to industry standards. It is the first time that FIA-MS/MS for metformin was developed surpassing all existing methods in terms of time of analysis. The quantification method was dependent on the formation of [M+H](+) using electrospray ionization (ESI) and employing multiple reaction monitoring (MRM) using quadrupole-linear ion trap (4000 QTRAP(®)) instrument. A deuterated internal standard (IS) of metformin bearing six deuterium atoms was used to compensate for matrix effects and for variation in ion current within the ESI source. The ion transitions that were monitored were m/z 130.1→m/z 71.0 and m/z 130.1→m/z 60.1 for metformin and m/z 136.0→m/z 77.0 for the internal standard. A linear response (r=0.9966) was established for a range of concentrations of 5-2340 ng/mL. The inter- and intra-day variations were within the acceptable criteria for all quality control samples. The method was successfully applied for measurement of serum metformin concentration in dogs after intravenous injection.