OBJECTIVE To establish a method for simultaneous determination of 20 flavonoid compounds from honey samples using solid phase extraction coupled with ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS), which was applied to study the difference of flavonoid compounds in honey samples from different botanical origins. METHODS The honey samples were extracted with ultra-water solution by ultrasound, and purified with an Oasis MAX column, and then separated on a Waters ACQUITY UPLC HSS T3(2. 1 mm×100 mm, 1. 8 μm) and eluted with the mobile phase consisted of 0. 1% formic acid in water and acetonitrile in a gradient program, and analyzed by mass spectrometry equipped with an electrospray ionization source, operating in the positive or negative ionization modes, and performed in the multiple reaction monitoring using external standards. RESULTS Twenty flavonoid compounds showed good linearity with coefficients higher than 0. 9969; The recoveries of spiked honey samples in the range of 76. 2%-108. 6% at the concentrations of 100, 250 and 500 μg/L; The relative standard deviations were 0. 50%-4. 34% and 2. 51%-6. 71%(n=6)for inter-day and intra-day precisions, respectively. It was found that the variations of flavonoid compounds in 16 types of honey samples from different botanical origins differed largely. Among them, quercetrin content in fennel honey was the highest(165 ng/g). High contents of luteolin in red eucalyptus and manuka honey were 186 ng/g and 304 ng/g, respectively. High contents of kaempferol in buckwheat, fennel and manuka honeys were from 113 to 257 ng/g. High contents of chrysin in snow lotus honey, manuka, sunflower honey and lavender Honey were from 136 to 213 ng/g. The content of pinocembrin(473 ng/g) and pinobanksin(684 ng/g) in manuka honey were much higher than that in other honeys. CONCLUSIONS The proposed method is simple for pretreatment, also sensitive, accurate and producible for quantitative of 20 flavonoid compounds in different honey samples, which provide more scientific data and application basis for quality analysis.