Du Zhong Formula (DZF), a traditional Chinese medicine formula derived from BeiJiQianJinYaoFang, is used to treat kidney deficiency and lumbago. In this study, Ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometer (UPLC-Q-TOF/MS) technique combined with pattern recognition analysis was applied for analysis of metabolic profiles of the bioactive components of the DZF in rat biological samples. In this experiment, a total of 73 compounds, including 53 prototype components and 20 metabolites, were identified tentatively in vivo compared with blank urine, plasma, feces and cerebrospinal fluid (CSF). The prototype ingredients in DZF include terpenoids, gingerols, phenylpropanoids, alkaloids, phenanthrenes, bibenzyls, organic acids and other ingredients. The metabolic pathways of DZF involved reduction, demethylation, hydroxylation, desugarization, deoxygenation, glucuronidation, sulfation, and methylation. The proposed method could develop an integrated template approach to analyze screening and identification of the bioactive components in plasma, urine, feces and CSF after oral administration of herb medicines. Additionally, this investigation might provide helpful chemical information for further pharmacology and activity mechanism of DZF.