Objective: To develop a method of high performance liquid chromatography coupled with a photodiode array detector(HPLC-DAD)for the content determination of six active flavonoids (quercetin-3-O-sambubioside

rutin and quercetin-3-O-β-D-glucopyranside

kaempferol-3-O-β-D-glucopyranside

quercetin

kaempferol)in Oldenlandia diffusa. Method: The analyses were performed on a Diamonsil-C18 column (4.6 mm×250 mm

5 μm) eluted with acetonitrile and 0.05% phosphoric acid in gradient mode. The flow rate was 1 mL·min-1

the detection wavelength was 254 nm

the column temperature was set at 30 ℃. Result: Six flavonoids have good linear relationship

precision

stability

and repeatability according to the requirements of the methodology determination. The recoveries were 99.40% -99.70%. Six flavonoids in O. diffusa from different origins were determined by HPLC-DAD. Conclusion: The method is simple

accurate

replicable

and can be used for the quality control of flavonoids in olden landia diffusa.