Abstract: Abstract ：The excretion of Vitamin B2 in human urine after oral administration of 50mg Vitamin B2 is studied. Urine samples in 1, 2, 4, 6, 8 hours were collected and extracted by solid-phase extraction. A HPLC method was established for determination of Vitamin B2 in human urine with mobil phase of acetonitrile/ ammonium buffer (0.01mol／L). The compounds was detected directly by ultraviolet detector at 267nm. This method is simply rapid, sensitive and cheap for directly determining Vitamin B2 in human urine with the recovery of 91.2%~96.7%.