<jats:title>Abstract</jats:title><jats:p>A highly sensitive and selective LC‐ESI‐MS was developed, validated for the simultaneous determination of 18<jats:italic>α</jats:italic>‐glycyrrhetic acid (<jats:italic>α</jats:italic>‐GA) and 18β‐glycyrrhetic acid (<jats:italic>β</jats:italic>‐GA) for pharmacokinetic studies in healthy subjects. Sample preparation was performed by liquid–liquid extraction with ethyl acetate and the separations were achieved using a C<jats:sub>18</jats:sub>column with the mobile phase composed of 10 mmol/L ammonium acetate solution–methanol–acetonitrile (40:36:24, v/v/v) at a flow rate of 1 mL/min. The internal standard was honokiol and the epimers were quantified using a single quadrupole mass spectrometer employing ESI in the negative ion mode. The separation factor,<jats:italic>α</jats:italic>, was 1.512 for<jats:italic>α</jats:italic>‐ and<jats:italic>β</jats:italic>‐GA. The standard curves were linear for both epimers with coefficients of determination (<jats:italic>r</jats:italic> ≥ 0.9998) over the concentration range of 1–150 ng/mL. The precision and accuracy were ≤ 4.33 and ≤ 6.57%, respectively. The mean plasma extraction recoveries were 82.23 ± 1.91 and 84.29 ± 2.09% for<jats:italic>α</jats:italic>‐GA and<jats:italic>β</jats:italic>‐GA, respectively. The assay was successfully applied to evaluation of the pharmacokinetic properties of<jats:italic>α</jats:italic>‐GA and<jats:italic>β</jats:italic>‐GA from 19 volunteers who had received oral administration of diammonium glycyrrhizinate capsules. The initial data suggest that glycyrrhizin metabolizes to glycyrrhetic acid fairly slowly and the elimination of<jats:italic>α</jats:italic>‐GA is slower than that of<jats:italic>β</jats:italic>‐GA. Copyright © 2008 John Wiley & Sons, Ltd.