The residue depletion of ampicillin was investigated in the olive flounder (Paralichthys olivaceus), rockfish (Sebastes schlegeli), and red sea bream (Pagrus major) after 5 days treatment with medicated feed at a dose of 100 mg/kg bw/day. Fishes were sampled for muscle on 1st, 2nd, 3rd, and 4th day after treatment. Ampicillin concentrations were determined by high performance liquid chromatography after SPE column extraction. The recovery rates of ampicillin in muscle samples ranged 94-98% and 83-88% for the concentration of 0.05 mg/kg and 0.1 mg/kg, respectively. Ampicillin concentrations detected on 1st day after treatment were 0.143, 0.138, and 0.187 mg/kg in the muscle of olive flounder, rockfish, and red sea bream, respectively. After a withdrawal of 3 days, muscle concentrations were 0.016, 0.012, and 0.021 mg/kg in the olive flounder, rockfish, and red sea bream, respectively. Ampicillin was not detectable in muscle samples on 4 days following withdrawal of the medicated feed. From results of the present study, a withdrawal period of ampicillin is proposed on 5 days after 5 days treatment with medicated feed at a dose of 100 mg/kg bw/day to avoid the presence of excessive residues of the edible muscles of olive flounder, rockfish, and red sea bream.