A method using liquid chromatography-electrospray ionization ion trap mass spectrometry was established for the identification of metabolites in feces, urine and bile in rats after oral administration of 3′,4′-dimethoxy flavonol-3-β-d-glucopyranoside (abbreviated DF3G). Seven metabolites in rat feces, urine and bile were firstly identified on the basis of their MS fragmentation behaviors. Three metabolites were identified in the feces, 6 in the urine and 2 in the bile, which suggested that demethylation, deglycosylation and deglycosylation followed by glucuronide conjugation were the major metabolic pathways for DF3G in vivo. Hydrolyzation might be the first step in the absorption and metabolism of DF3G. The possible metabolic pathway was proposed for the first time. The established method was simple, reliable and sensitive, revealing that it could be used to rapidly screen and identify the structures of metabolites of DF3G to better understand its metabolism in vivo.