Ziziphi spinosae semen (ZSS) is a traditional Chinese medicine that has been widely used to treat insomnia and anxiety. Modern pharmacological studies have demonstrated that flavonoids are the main active compounds in ZSS. However, the metabolites and the metabolic pathways of flavonoids in ZSS have not been investigated thoroughly. In this study, a method based on high-performance liquid chromatography coupled with Fourier transform ion cyclotron resonance mass spectrometry (HPLC/FTICR-MS) was established to identify the metabolites of flavonoids after oral administration of extract of ZSS to rats or dogs, using parent mass list-triggered data-dependent multiple-stage mass analysis at a resolving power of 50,000 in the external calibration mode. The mass accuracies obtained for all full-scan analyses were less than 4 ppm (<2 ppm in most cases). A total of 15 compounds were detected in biological samples of rats and dogs, and nine compounds were identified. The metabolic pathways of flavonoids of ZSS in rats and dogs were proposed. The results may help better understand the material basis and pharmacological mechanism of ZSS. Copyright © 2012 John Wiley & Sons, Ltd.