OBJECTIVE： To establish the method for the simultaneous determination of 4 flavonoids in rat plasma sample， such as quercetin-3-O-sophoroside， isoquercitrin， hyperin， rutin. METHODS： 8 SD rats were selected and given Poacynum hendersonii leaf extract suspension intragastrically 0.1 g/kg （calculated by extract）. The blood sample 1 mL was collected from orbit 1 h after medication. The contents of 4 flavonoids were determined by microemulsion liquid chromatography （MELC） after centrifugation. The separation was performed on Zorbax SB-C18 column with mobile phase consisted of microemulsion [polyoxyethylene lauryl ether-n-butanol-ethyl acetate-triethylamine-water （volume ratio was 2.5 ∶ 3.0 ∶ 1.7 ∶ 0.3 ∶ 92.5， pH＝5.0）] at the flow rate of 0.8  mL/min. The column temperature was set at 30 ℃， and detection wavelength was 360 nm. Sample size was 10 μL， and internal standard was bergenin. It was compared with HPLC method using organic solvent as mobile phase （mobile phase consisted of 0.2％ phosphoric acid solution-acetonitrile， gradient elution， other condition same as MELC method）. RESULTS： In rats plasma， the linear range of quercetin-3-O-sophoroside， isoquercitrin， hyperin， rutin were 10-1 000 ng/mL （r≥0.998 0）. The limits of quantitation was 10 ng/mL （S/N＝10）. RSDs of precision，stability and reproducibility tests were all below 7.5％ （n＝5）. Average sample recoveries were between 97.4％-98.5％ and RSDs were less than 5.3％ （n＝5）. Extraction recoveries were between 88.7％-100.3％ （RSD≤6.14％， n＝5）. The methodological evaluation results of MELC and HPLC method were all in line with the regulations of pharmacopeia， and the results of blood concentration between them were similar. Compared with HPLC method， MELC method could shorten detection time （10 min vs. 40 min） and reduce the amount of organic solvent （0.38 mL vs. 28 mL）. CONCLUSIONS： Established MELC method is rapid， simple and green， and can be used for 4 flavonoids from P. hendersonii leaf in rat plasma sample.