Folic acid is an essential nutrient, and folate deficiency is associated with a variety of disorders including neural tube defects (during pregnancy) and heart disease. A fast, sensitive, and robust HPLC-tandem mass spectrometry (LC-MS-MS) method was developed for the quantification of free folic acid, tetrahydrofolate, 5'-methyltetrahydrofolate, and 5'-formyltetrahydrofolate in human plasma. Sample preparation required only acetonitrile precipitation of proteins followed by filtration instead of solid-phase extraction or solvent-solvent extraction as in other methods. The rapid and streamlined sample handling procedure minimized degradation of the highly unstable folate species. Hydrophilic interaction chromatography was used for additional sample cleanup on-line, and baseline separation and detection of all four folate species was achieved in less than 30 min. The folate species were detected using negative ion electrospray-tandem mass spectrometry with multiple reaction monitoring of the diagnostic fragment ions of each deprotonated molecule. The predominately organic (hydrophobic) solvent system combined with the microbore flow rate (50 L/min) used for the chromatography resulted in enhanced electrospray signal response compared to reversed-phase HPLC using a wider bore column. The recovery of all folate species (from spiked plasma) was >97␘ver a concentration range from 300 pg/L to 12 mg/L with intraday precision (RSD, n = 5) of 3.7-6.5ÐStability studies were carried out for spiked samples in order to define storage and handling conditions. The folic acid limit of quantification (LOQ) in human plasma was 80 pmol/L ± 10°and the limit of detection (LOD) was 37.5 pmol/L. The LOQ and LOD for tetrahydrofolate, 5'-methyltetrahydrofolate, and 5'-formyltetrahydrofolate were 1250, 400, and 360 pmol/L of plasma and 425, 165, and 140 pmol/L of plasma, respectively.