In this study, the pharmacokinetic profiles of the bioactive components in the leaf extract of the medicinal herb, Cudrania tricuspidate , were investigated using an MS/MS-based molecular networking system. To identify the major active components of the C. tricuspidate leaf extract (CLE), HPLC-DAD analysis was conducted with a standard mixture of six flavonoids (rutin, isoquercitrin, nicotiflorin, kaempferol 3-O-glucoside, quercetin, and kaempferol). The unknown peaks were determined via molecular networking analysis using the mass dataset obtained by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF/MS). For the subsequent pharmacokinetic study, CLE (1 g/kg) was orally administered to rats, and plasma samples were collected. The product ion mass data of plasma samples using LC-QTOF/MS were obtained and subjected to molecular networking analysis. The resulting molecular networking map indicated that the glucuronide metabolites of quercetin and kaempferol were the major circulating species. Accordingly, quercetin and kaempferol were determined following β-glucuronidase treatment, and their pharmacokinetic parameters were calculated. These findings indicate that the proposed molecular network-based approaches are potential and efficient methods for the pharmacokinetic study of herbal medicines.