An LC-MS/MS method has been developed for the determination of main monoglutamate folates in spinach with folic acid as an internal standard. A sample preparation with ultrafiltration (molecular weight cut-off membrane, 5 kDa) was followed by a chromatographic run of 14.2 min, rendering the method very simple and fast. The LODs in diluted spinach matrix were 0.02, 0.09, 0.05 and 0.03 ng/mL (0.037, 0.17, 0.092 and 0.055 microg/100 g calculated according to the fresh weight of spinach) for 5-methyltetrahydrofolate, tetrahydrofolate, 5-formyltetrahydrofolate, and 10-formylfolic acid, respectively. Using this method, the extraction behaviour of the main naturally occurring monoglutamate folates has been investigated in detail. It is found that 10 min of heating at 100 degrees C, incubation with rat serum at 37 degrees C (0.05 M phosphate buffer, pH = 6.5) for 4 h and the ratio of 10 (volume of extraction buffer versus the weight of sample, mL/g) are the optimal parameters for folate extraction from spinach. The final quantitative result of the individual folates in spinach is highly influenced by the pH (from 2.9 to 8.6) of the extraction buffer.