The Chinese herbal formula, Shu-Yu capsule (SYC), has been successfully used to treat depression-like disorders in clinical settings. To rapidly identify the chemical constituents of SYC and its metabolites in rat serum, a simple and sensitive liquid chromatography-tandem mass spectrometry method was established in the present study. By comparing the retention times, MS and MSn spectra data in the literature and reference standards, a total of 73 compounds were identified from SYC. In rat serum, 62 components, including 13 prototype compounds and 49 metabolites were identified. Of these components, 14 metabolites were confirmed as novel metabolites of SYC. The results of the present study indicated that certain flavonoid glycosides and monoterpene glycosides were absorbed directly. Glucuronidation and sulfation were identified as the predominant metabolic pathways of the components in SYC. In addition, certain phase I reactions, including hydrolysis, demethylation and hydroxylation occurred in the rats. These results provide scientific evidence, which support further investigations of the pharmacology and mechanism of SYC.