Abstract: In order to study the determination method and optimum extraction process of two flavonoids from Ixeris polycephala, the Lactuca indica L.cv.Mengzao was used for research material. The content of rutin and quercetin were determinated by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry, meanwhile, the detection menthod was established. This paper adopted the technology of ultrasonic-assisted ethanol extraction, designed single factors and L₉（3~4） orthogonal test to optimize the process conditions, including solid-liquid ratio, ethanol volume fraction, extraction time and extraction temperature. The results showed that the linear relationship between rutin and quercetin was good in the linear range of 0.03-1 μg/mL, the correlation coefficient（R~2） was greater than 0.999. The average recoveries of rutin and quercetin was 98.59% and 96.81%, and RSD was 2.69% and 3.32%, respectively. The optimized extraction conditions were as follows: the solid-liquid ratio was 1∶15（g/mL）, the ethanol volume fraction was 60%, the extraction time was 30 min and the extraction temperature was 70 ℃. Under these conditions, the extraction content of rutin and quercetin from the leaves of Lactuca indica L. cv.Mengzao was 78.85 μg/g and 18.66 μg/g, respectively. The detection method of ultra performance liquid chromatography-mass spectrometry was established, and rutin and quercetin were extracted from Lactuca indica L. cv.Mengzao in this study. This method was fast, reliable, simple and accurate, and it could be used for the extraction and determination of flavonoids in the Ixeris polycephala.