Abstract: OBJECTIVE To study the metabolites of 5-hydroxyflavone in rat. METHODS High performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) was used for analysis of metabolites of 5-hydroxyflavone in rat after oral administration. The separation was performed with a Zorbax C18 column using a binary gradient elution of 0.05% formic acid acetonitrile-0.05% formic acid water. The quasi-molecular ions of compounds in positive mode were observed for molecule mass information, and the potential structures were presumed. RESULTS Parent component 5-hydroxyflavone was found only in rat fence, while glucuronide conjugates of 5-hydroxyflavone were found in rat urine, fence, plasma and bile. CONCLUSION Absorption of 5-hydroxyflavone is poor, and the major forms of 5-hydroxyflavone in rat are metabolites of phase II glucuronide conjugates.