<jats:sec><jats:title>RATIONALE</jats:title><jats:p>Seeds of <jats:italic>Vaccaria segetalis</jats:italic> (<jats:italic>Wang‐Bu‐Liu‐Xing</jats:italic> in Chinese) are mainly used in traditional Chinese medicine for the treatment of amenorrhea, breast infections, and edema. The study was designed to identify the components and metabolites of <jats:italic>Wang‐Bu‐Liu‐Xing</jats:italic>.</jats:sec><jats:sec><jats:title>METHODS</jats:title><jats:p>A novel methodology combining an adsorbent‐separation strategy with analysis by liquid chromatography/quadrupole time‐of‐flight tandem mass spectrometry (LC/QTOF‐MS/MS) was established to identify the components of <jats:italic>Wang‐Bu‐Liu‐Xing</jats:italic>. The adsorbent‐separation technique was applied on macroporous resin (adsorbents). Different concentrations of ethanol (30%, 60%, and 95%), which covered high‐to‐low polarity ranges, were chosen as the elution solvent, respectively. The QTOF mass spectrometer was operated in negative ion mode with an electrospray ionization source.</jats:sec><jats:sec><jats:title>RESULTS</jats:title><jats:p>A total of 52 components were successfully identified in the <jats:italic>Wang‐Bu‐Liu‐Xing</jats:italic> decoction based on the fragmentation pathways and QTOF high‐accuracy mass spectral analysis. To the best of our knowledge, several new saponins were reported for the first time. A total of 20 compounds, which included 10 prototypes and 10 metabolites, were also identified in rat plasma and urine after oral administration of <jats:italic>Wang‐Bu‐Liu‐Xing</jats:italic> decoction.</jats:sec><jats:sec><jats:title>CONCLUSIONS</jats:title><jats:p>An integrated adsorbent‐separation strategy is powerful and reliable for global detection and identification of complex components in herbal prescriptions. The components identified in rat biofluids may also provide helpful chemical information for further pharmacology and active mechanism study on this herb. Copyright © 2014 John Wiley & Sons, Ltd.</jats:sec>