Abstract: Objective: To establish methods for qualitative and quantitative analysis of isorhamnetin and content determination of total flavonoids from pollen of Paeonia rockii.Methods: The identification of isorhamnetin from pollen of Paeonia rockii was performed by TLC.The content of isorhamnetin was determined by HPLC.Agilent Eclipse plus C18column ( 250 mm × 4.6 mm, 5 μm) was adopted, the mobile phase was methanol-acetonitrile-0.1% phosphoric acid ( 25 ∶ 13 ∶ 62) , at the flow rate of1.0 m L·min-1. The detection wavelength was 360 nm. The content of total flavonoids was determined by UV-visible spectrophotometry.Results: The TLC method was suitable for the identification of isorhamnetin with good reproducibility. The linear of isorhamnetin was in the range of 8.4 ~ 168.0 μg·m L-1 ( r = 0.9999) , and the average recovery ( n = 9) was 100.8% ( RSD = 2.36%) , the content of isorhamnetin from pollen of Paeonia rockii was 3.21⁸.91 mg·g^-1.The linear range of total flavonoids was 0.01⁰.06 mg·m L-1 ( r = 0.9992) and the average recovery ( n = 9) was 101.06% ( RSD = 2.56%) , the content of flavonoids from pollen of Paeonia rockii was 9.44¹5.96 mg·g^-1.Conclusion: The developed methods are simple, accurate and suitable for the quality control of pollen of Paeonia rockii.