A new, simple, specific gas chromatography-mass spectrometry method (GC-MS) was developed for the determination of metformin in human plasma. A number of derivatization approaches (silylation, acylation and methylation) were tested to derivatize metformin prior to GC-MS analysis. Derivatization of metformin was achieved by using N-methyl bis(trifluoroacetamide) (MBTFA). Several parameters such as reaction temperature and time were optimized, which affected the yield of the derivatization reaction. A trifluoroacetyl derivative of metformin was identified and quantified in selected ion monitoring mode (mass-to-charge ratio (m/z): 303). The method was fully validated using parameters such as specificity, carryover, linearity, limits of detection and quantification, precision, accuracy, stability, recovery, robustness and ruggedness. Linearity was demonstrated over the concentration range of 100-3000 ng mL(-1) with a coefficient of determination (R-2) above 0.996. The limits of quantification and detection were found to be 100 and 40 ng mL(-1), respectively. Intra- and inter-day accuracy and precision were within the acceptable limits (<15% for concentration points in the calibration range; <20% for the limit of quantitation). The developed method was successfully applied for the identification and quantification of metformin in the plasma of diabetic patients.