There are two purposes in this research, one is to develop the new method which can be used for detection and quantification of chloramphenicol in fish, and the method is according to Commission Decision 2002/657/EC. The other is to study about the depletion of chloramphenicol in Tilapia which is the main aquacultural product in Taiwan. Homogenized fish tissues were extracted with EtOAc and defatted with Hexane. HPLC separation was conducted on RP18 column in 0.5mM ammonium acetate ¡V MeOH. Chloramphenicol was determinated by LC-ESI-MS-MS in negative mode. The recoveries of chloramphenicol from fish gill, gut, liver and muscle were 68.8, 73.3, 82.7 and 85.5%. The limit of detection was 0.05, 0.14, 0.07 and 0.03 ng/g. Decision limit(CC£\) of fish muscle was 0.05 ng/g and detection capability(CC£]) was 0.07 ng/g. Chloramphenicol was administered by oral and by bathing respectively in Tilapia. Chloramphenicol was given by bathing at 45.8 mg/kg for one hour. No chloramphenicol was demonstrated in gill, gut and fish muscle on 193th hour, 673th hour, and 385th hour respectively after the beginning of this study. There was 0.08 ng/g chloramphenicol in liver after 385th hour. Chloramphenicol was given orally to Tilapia at 354.5 mg/kg daily for 3 days. No chloramphenicol demonstrated in fish muscle on 480th hour. There were 0.09 ng/g and 0.16 ng/g in liver and gut respectively. No matter oral experiment or bathing experiment, th concertration of chloramphenicol in liver is higher than in the other, the depletion rate in high concentration was faster than in low concentration.