Abstract: OBJECTIVE To establish an analytical method for simultaneous determination of quercetin, galangin and chrysin in Populus canadensis Moench. leaves. METHODS The determination was performed on Agilent TC C₁₈ column (4.6 mm×250 mm, 5 μm) with the mobile phase consists of methanol-0.1% formic acid at a flow rate of 1.0 mL·min^-1. The following gradient elution was used: the content of methanol was 25% to 55% from 0 to 5 min, 55% to 65% from 5 to 25 min and 65% to 75% from 25 to 35 min. The detection wavelength was set at 264 nm, and the column temperature was 30 ℃. RESULTS This method had a good separation performance for quercetin, galangin and chrysin in poplar leaves, and the linear range were 3.7-120, 2.5-80 and 3.125-100 μg·mL^-1, respectively; the average recovery rates were 99%, 100.1% and 101.4%, respectively. CONCLUSION This method is accurate, reliable with good stability and repeatability which provides the evidence for quality evaluation for poplar leaves.