Pterostilbene, a stilbene phytoalexin, is mainly obtained from blueberries and grape vines; however, its metabolic mechanisms were unclear in vivo. In the present study, three different methods were used to prepare biological samples, and then, an efficient strategy based on ultrahigh-performance liquid chromatography coupled with mass spectrometry was developed to screen and identify pterostilbene metabolites in rat urine, plasma, liver, and feces. In order to elucidate pterostilbene or its metabolites involved in vitro, this study was assessed by the liver microsome system. As a result, a total of 88 pterostilbene metabolites were characterized. Among them, 77 metabolites in vivo and 14 metabolites in vitro were found; 50 and 38 metabolites were observed in rat plasma and urine, while only 4 and 12 metabolites were detected in rat feces and liver, inferring that plasma and urine possessed more diverse types of pterostilbene metabolites; 41 metabolic products were obtained by solid-phase extraction, and 9 and 10 metabolites were screened by methanol precipitation and acetonitrile precipitation, respectively, indicating that solid-phase extraction could be adopted as the most acceptable method for pterostilbene metabolism. The results also demonstrated that pterostilbene mainly underwent glucosylation, dehydrogenation, hydrogenation, demethoxylation, sulfation, NAC binding, methylene ketogenic, acetylation, and methylation. In summary, this research provides an idea for the further study of drug metabolism.