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A novel LC–MS/MS method was developed for enantioselective separation of Schisandrin B on a Chiralpak IC column.The method was successfully used in investigating the stereoselective pharmacokinetics of Schisandrin B.The presented method is simple and reproducible.The validation of enantioselective behavior of Schisandrin B provides a reference for clinical practice.Schisandrin B (Sch B) has received much attention owing to its various biological activities. Schisandrin B exists as a racemate in “wuweizi”, a traditional Chinese medicine in China. In the present study, a novel chiral LC–MS/MS method was developed for enantioselective separation and determination of Schisandrin B in rat plasma. The plasma samples were prepared by liquid-liquid extraction (LLE). Schisandrol B was used as internal standard. Chiral separation was obtained on a Chiralpak IC column using 0.1% (v/v) formic acid in mixture of methanol and water (90:10, v/v) as a mobile phase. Parameters including the selectivity, linearity, precision, accuracy, extraction recovery, matrix effect and stability were evaluated. The method described here is simple and reproducible. The lower limit of quantification of 5.0 ng/mL for each Sch B enantiomer permits the use of the method in investigating the stereoselective pharmacokinetics of Sch B. Following racemic Sch B and “wuweizi” extracts, the area under the curve of (8R, 8′S)-Sch B was statistically higher than the one of (8S, 8′ R)-Sch B, with a ratio of 1.16–1.40 in three cases. This study firstly reports the development and validation of enantioselective behavior of Sch B in vivo, and provides a reference for clinical practice and encourages further research into Sch B enantioselective metabolism and drug interactions.