A rapid and sensitive liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method was developed and validated for determining protostemonine, a new antitussive agent isolated from Radix Stemonae. Separation was performed on a C-18 column with mass detection in positive selected reaction monitoring mode at the transitions of m/z 418.2 -> m/z 320.2 and m/z 416.2 -> m/z 342.2 for protostemonine and internal standard, respectively. The assay showed good linearity (r > 0.998) over the tested concentration range with the lowest limit of quantification of 1.0 ng/ml. The intra- and inter-day precisions (RSD, %) were 2.21-9.89% and 3.99-13.19%, respectively; whereas accuracy (RR, %) ranged from 90.35% to 108.32%. The extraction recovery, stability, and matrix effect were demonstrated to be within the acceptable limits. The validated assay was further successfully applied to the pharmacokinetic studies of protostemonine in rat. Protostemonine was rapidly eliminated from plasma following single intravenous administration (2 mg/kg) with a t(1/2) of 3.06 +/- 1.37 h. After oral administration (10, 20, and 50 mg/kg), protostemonine was rapidly absorbed from the gastrointestinal tract with t(max) of approximately 1 h, and has shown dose-independent pharmacokinetic behaviors. Oral bioavailability of protostemonine was calculated to be 5.87-7.38%. Moreover, a total of 10 metabolites were structurally identified by using UHPLC-QJTOF-MS method. The proposed metabolic pathways of protostemonine in rat involve demethylation, hydrolysis, and oxygenation. The current study provides informative data for understanding the in vivo disposition of protostemonine, which, in turn, help in interpreting the mechanism of its effectiveness and toxicity. (C) 2015 Elsevier B.V. All rights reserved.