In this study, a LC/MS/MS method for the determination of ampicillin antibiotic in fish muscle tissue was developed and accredited according to Commission Decision 2002/657/EC. The metabolism of ampicillin in tilapia was them studied in serum, liver and muscle. The homogenized fish tissue was first extracted with MeOH-H2O(4:1), C18 sorbent was added to remove lipids and impurities, the extract was then evaporated to dryness with a steam of nitrogen gas at 38 ¢XC. The residue was redissolved with H2O, filtered and analyzed by LC/MS/MS equipped with an Agilient HC-C18(5£gm, 150mm ¡Ñ4.6mm), the mobile phase A was 10mM ammonium acetate containing 0.1% formic acid, while the mobile phase B was methanol. The determination of ampicillin was performed with electrospray ionization-tandem mass spectrometry in positive mode using multiple reation monitoring(MRM) for detection. Average recoveries were 81¡V86%, the limit of detection was 6.00 £gg kg-1¡Adecision limit(CC£\) of ampicillin in fish muscle sample was 63.65 ¡Ó 7.99 £gg kg-1. In the metabolism study, the oral administered dose to talipia was 20 mg/kg¡DBW. The maximum concentration of ampicillin in each tissues was obserned at 0.5 hour after oral administration, the maximum concentration in serum, liver and muscle was 27.53 mg L-1, 66.75 mg kg-1 and 1.33 mg kg-1, respectively. The concentration of ampicillin in muscle was 0.04 mg kg-1 24 hours after oral administration, which is lower than the 0.05 mg kg-1 MRL value of European Union resolutions. No residual ampicillin was detected in tilapia 48 hours after oral treatment, which conformed to the drug regulations for aquaculture ainmals in Taiwan.