Hydroxymethylglutaryl coenzyme A reductase (HMGCR) catalyzes the rate limiting step in cholesterol biosynthesis converting HMG-CoA into mevalonic acid (MVA), which equilibrates with mevalonic acid lactone (MVL) under neutral pH conditions. We developed a fast, sensitive, and efficient method to determine HMGCR activity in human cell lines measuring MVL levels by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Convenient prepared samples containing MVL-D-7 as an internal standard were injected, separated, and eluted from an ACQUITY HSS PFP column. Measurement of MVL was performed by electro-spray ionization mass spectrometry with multiple reaction monitoring. Calibration curves were linear and reproducible in the range of 0.15-165 mu g/l (r > 0.99). Lower limit of quantification was 0.12 mu g/l. Intra- and interassay imprecision were <1.3% and <2.9%, respectively. HMGCR enzymatic activity measurements of cells cultivated under different cell culture conditions (with 10% FCS, with 10% lipoprotein-deficient serum and under serum starvation) revealed the applicability of this test system for various experimental settings. This efficient UPLC-MS/MS assay permits rapid and high sensitive determination of HMGCR enzyme activity, tracing potential alterations in cholesterol biosynthesis. (C) 2013 Elsevier Inc. All rights reserved.