To establish a method for the simultaneous determination of total flavonoids in 12 different samples of Lepidium latifolium L.by ultraviolet spectrophotometry.AlCl₃ colorimetry was used to determine the content of total flavonoids in 12 samples of L. latifolium from different locations with the detection wavelength of 421 nm and rutin as the control.The results showed that the total flavonoids had a good linear relationship (r=0.998).The average recovery of sample addition is 96.77%,which ranged from 91.83%~102.50%,with RSD 3.39%.The method is simple,accurate and reproducible,and can be used as a reference for quality control of L. latifolium.