Abstract: A 4th order derivative UV spectrophotometric method was developed for the determination of two major metabolites (TFu and 5-Fu) of A-OT-Fu in mice serum following per os of 290 mg/kg of the drug. TF u and 5-Fu were extracted from mice serum by ethyl acetate The amplitudes of 252 and 289 nm for axle are linear with concentration of TFu and 5-Fu within the range of 5～50μg/ml. Under the given experimental condition the recovery of TFu and 5-Fu were over 95%. The maximum concentration of TFu and 5-Fu appeared at 1 h and 2 h respectively after administration.