Abstract: For further study of bioactive components of cryptotanshinone in vivo, the metabolites of cryptotanshinone in rats were analyzed and identified by ultra-high performance liquid chromatography coupled with quadrupole/electrostatic field Obitrap high-resolution mass spectrometry (UHPLC-Q-Exactive MS). Cryptotanshinone was suspended in 0.2% Tween-80 solution. Rats in drug group were given adose of 300 mg/kg body weight orally. 0.2% Tween-80 solution was administrated to rats in control group. Plasma at different time points was collected after oral gavage. Urine and feces were collected after single intragastric administration in rats within 24 h. Biological samples (plasma, urine and feces) were separated by solid phase extraction (SPE) to exclude protein and solid residue precipitation. Acquity UPLC BEH C18 column (2.1 mm×100 mm×1.7 μm) was used with 0.1% formic acid solution (A)-acetonitrile (B) as the mobile phase for the gradient elution. Then the biological sample was analyzed by quadrupole/electrostatic field Obitrap high-resolution mass spectrometry at positive ion mode. According to accurate molecular weight, primary and secondary mass spectrometry information and literatural reports, a total of 45 metabolites (including cryptotanshinone) were screened and identified. The main metabolic of pathways in rats are reduction, hydroxylation, demethylation, glucuronidation, S-cysteine acid binding reaction, decarbonation, dehydrogenation, deethylation, deacetaldehydeation and their composite reactions. This study not only provides useful data to comprehensively understand the metabolic mechanism and material basis of cryptotanshinone, but also showes that it is reliable for further study of drug-related constituents and quality standard of cryptotanshinone.