To determine the content of five kinds of flavonoids in the seed of semen cuscutae, including hyperoside, astragalin, quercetin， kaempferol and isorhamnetin. Methods： The HPLC method was used to determine the content of flavonoids in the seeds of semen cuscutae. The analysis was carried out on Agilent TC C18 column (250 mm×4. 6 mm, 5 μm), with MeOH-0. 4 %H3PO4as mobile phase, the flow rate of 1 mL·min-1 and wave length at 360 nm. The gradient elute time was 0→60min→80min, and the proportion of methanol was 25%→40%→60%.at45℃. Results： Chromatographic peak separation was in good condition, and five kinds of flavonoids had good linear relationship respectively within their own scope of concentration; the recovery was 98.0% ～ 98.0%, RSD was 1.44% ～2.63%. Conclusion：The established method is simple, accurate, and reproducible and can be used as the quality evaluation of semen cuscutae.