The use of high-performance liquid chromatography for the determination of folates is well documented. The methods used are based on reversed-phase chromatography with UV and/or fluorescence detection. In many instances it is difficult to reach the required detection limits and many of the methods lack specificity. High-performance liquid chromatography-mass spectrometry (LC-MS) has become a well established analytical tool in modern laboratories. It can offer superior specificity and often lower detection limits than conventional LC detection methods and thus is ideally suited to the analysis of folates. A system capable of separating the four main folates [folic acid (pteroylglutamic acid, PGA)], 5-methyltetrahydrofolic acid, terahydrofolic acid and 5- and/or 10-formyltetrahydrofolic acid [folinic acid (CHOTHF)] using LC-MS with negative ion electrospray has been developed. After optimisation, a system using a 12.5 cm x 3 mm, 3 microm Hypersil ODS column and a mobile phase of 2.5 mM acetic acid-acetonitrile (88:12, v/v) was developed which was capable of separating the four folates in under 10 min without the use of a gradient system. Extraction of the folates is by heat treatment and sample clean-up is by solid-phase extraction (C18). On column limits of confirmation are 0.6 ng for PGA and CHOTHF.