OBJECTIVE： To establish a method for simultaneous determination of glucoraphanin and 4-methylsulfoyl-3-butenyl glucosinolate in the seeds of Raphanus sativus. METHODS： HPLC was adopted. The determination was performed on Welch Ultimate LP-C18 column with mobile phase consisted of methanol-0.05 mol/L monobasic potassium phosphate （gradient elution） at the flow rate of 1.0 mL/min. The detection wavelength was set at 235 nm， and column temperature was 30 ℃. The sample size was 20 μL. The established method was used to determine the contents of glucoraphanin and 4-methylsulfoyl-3-butenyl glucosinolate in 15 batches of samples from 5 producing areas. RESULTS： The linear range of glucoraphanin and 4-methylsulfoyl-3-butenyl glucosinolate were 0.248-3.960 and 0.903-14.442 μg （r≥0.999 6）， respectively. The detection limits were 0.009 9 and 0.003 5 μg， and limits of quantitation were 0.024 8 and 0.008 9 μg， respectively. RSDs of precision， stability （24 h） and reproducibility tests were all lower than 2％ （n＝6）. The average recoveries were 99.83％ and 100.40％ （RSD＝1.42％， 1.50％，n＝6）. The average contents of 15 batches of glucoraphenin in the seeds of R. sativus from producing areas （Anhui， Gansu， Hebei， Shandong， Yunnan） were 1.881， 5.120， 3.634， 3.237 and 2.856 mg/g （n＝3）. Average contents of 4-methylsulfoyl-3-butenyl glucosinolate were 37.561， 46.389， 40.136， 37.000， 41.101 mg/g （n＝3）. CONCLUSIONS： The established method is simple， accurate and repeatable， which can be used for content determination of glucoraphanin and 4-methylsulfoyl-3-butenyl glucosinolate in the seeds of R. sativus. Among samples of 5 producing areas， average contents of glucoraphanin and 4-methylsulfoyl-3-butenyl glucosinolate in the seed of R. sativus from Gansu province are the highest.