A simple plasma extraction method coupled with liquid chromatography–tandem mass spectrometry (LC/MS/MS) detection was developed and validated for the analysis of endogenous mevalonic acid (MVA), a biomarker indicative of the rate of cholesterol biosynthesis, in human plasma samples. The analyte was extracted from the plasma matrix using a straightforward liquid–liquid sample preparation procedure. The extract supernatants were evaporated, reconstituted in aqueous solvent and injected into the LC/MS/MS system without further processing. The chromatographic separation was achieved on a reverse‐phase high‐performance liquid chromatography column. The accuracy and precision of the method was determined over the concentration range 0.25–25 ng/mL MVA from human plasma extracts in three validation batch runs. Inter‐assay precision (%CV) and accuracy (%RE) of the quality control samples were ≤7.00% (at lower limit quality control) and ≤6.10%, respectively. The sensitivity and throughput of this assay was significantly improved relative to previously published methods, resulting in smaller sample requirements and shorter analysis time. Assay results from a clinical study following the oral administration of an exploratory statin demonstrate that this procedure could potentially be used in the investigation of therapies associated with hypercholesterolemia. Copyright © 2010 John Wiley & Sons, Ltd.