Folates are important cofactors in one-carbon metabolism. Disturbances in folate homeostasis and metabolism may be related to an increased risk of cardiovascular disease and carcinogenesis and may lead to congenital malformations, namely neural tube defects. Determination of these compounds in biological samples is often a problem due to the existence of numerous folate metabolites, their relative instability, and the low contents in serum and most tissues. As existing methods have distinct limitations, we developed a method, which facilitates the separation as well as the sensitive detection of eight folates by coupling HPLC with a microbiological assay. After a simple sample preparation, including deproteinization and enzymatic hydrolysis of folate polyglutamates, extracts were chromatographed, fractions were collected on microtiter plates, and folates were quantitated using the Lactobacillus casei assay. The raw data were processed using a computing system after reconstructing the HPLC chromatogram with the bacterial growth data. Using the described method, the eight physiologically occurring folate monoglutamates could be simultaneously determined. The detection limits were 2-20 fmol per injection. The application of the method was demonstrated with the analysis of the folate pattern in milligram or sub-milligram quantities of plasma, erythrocyte, and embryos of pregnant mice during organogenesis.