Abstract: OBJECTIVE To develop an UPLC-MS/MS method for simultaneously determination of six flavonoids(rutin, hyperoside, quercetin-3-O-glucuronide, isoquercitrin, quercitrin, quercetin) in Cyclocarya paliurus. METHODS The UPLC method was performed on an Agilent ZORBAX RRHD SB-C₁₈ column(2.1 mm×100 mm, 1.8 μm) with a gradient elution of acetonitrile and 0.1% formic acid in water at a flow rate of 0.3 mL·min^-1, the column temperature was 40℃, and the injection volume was 1 μL; mass spectrometry used the triple quadrupole mass spectrometry, electrospray ionization source, negative mode(ESI^-) and the multiple reaction monitoring mode was employed for the quantitative analysis of six flavonoids. RESULTS The linear ranges of rutin, hyperoside, quercetin-3-O-glucuronide, isoquercitrin, quercitrin and quercetin were 0.002-0.24 μg·mL^-1 (R²=0.999 4), 0.049-4.91 μg·mL^-1(R²=0.999 5), 0.504-50.37 μg·mL^-1(R²=0.999 4), 0.051-5.12 μg·mL^-1(R²=0.999 4), 0.005-0.52 μg·mL^-1(R²=0.999 8), 0.052-5.17 μg·mL^-1(R²=0.999 9); the average recoveries were 96.1%, 98.4%, 98.5%, 97.3%, 96.7%, 96.5%, respectively. The RSDs were 1.78%, 1.22%, 1.91%, 1.08%, 1.21%, 1.29%, respectively. The content of six flavonoids in 16 batches were 0.019 8-0.253 7, 0.923 5^-15.568 0, 12.987 8^-130.510 9, 0.122 9^-12.101 1, 0.007 4-0.573 9, 0.387 7^-12.456 7 mg·g^-1, respectively, and the content of quercetin-3-O-glucuronide was the highest, which accounted for about 78% of the total amount. CONCLUSION The developed method is simple, rapid, sensitive and specific, which can be used for the determination of flavonoids and the quality control of Cyclocarya paliurus.