Abstract: A sensitive and specific HPLC-MS/MS method was developed for the quantitative determination of folic acid and 5-methyltetrahydrofolic acid in human plasma. Plasma samples were prepared through SPE cartridges under subdued laboratory light environment and separated through an Waters XTerra MS C18 column with methanol: 5mmol/L ammonium: acetate (pH6.0) = 25∶75 (v/v) as the mobile phase. Analytes were ionized in the gas-auxiliary electrospray ionization source (ESI) and positive ions were detected by mass analyzer in multiple reaction monitoring mode. Then peak areas were quantitated. The results showed both of the limits of quantitation of folic acid and 5-methyltetrahydrofolic acid were 0.5ng/mL, and the linear ranges were 0.5—25ng/mL. Correlation coefficients of all stadard curve are r≥0.99. Accuracies and precisions of this method are within ±15%. This method is specific, sensitive and accurate and will be applied to the determination of folic acid and 5-methyltetrahydrofolic acid in human plasma.