Nevadensin is major constituents of Lysionotus pauciflorus Maxim. (Chinese name: Shidiaolan), which has a variety of pharmacological effects such as anti-mycobacterium tuberculosis activities, antitussive, anti-inflammatory and anti-hypertensive. In this paper, we investigated the metabolism of nevadensin in vitro and in vivo. A strategy was firstly developed to identify the metabolites of nevadensin by using ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS). An on-line data acquisition method a multiple mass defect filter (MMDF) combined with dynamic background subtraction (DBS) was developed to trace all probable metabolites. Furthermore, some assistant tools, such as key fragment ions (KFI), were employed for compound hunting and identification. Based on the proposed method, 23 metabolites were structurally characterized in vivo including 16 phase I and 7 phase II metabolites, and 12 metabolites were detected in vitro containing 10 phase I and 2 phase II metabolites. The results indicated that oxidation, hydrolysis, demethylation, methylation, sulfate conjugation and glucuronide conjugation were main metabolic pathways of nevadensin. In a word, this study maybe can provide reference and valuable evidence for further investigation of the metabolic mechanism of nevadensin.