Florfenicol, a derivative of thiamphenicol in which the hydroxyl group at C-3 has been replaced with fluorine, is listed by the World Health Organization as an antibacterial agent for human medicine that is critically important in risk management strategies for non-human use. AOAC International has also identified it as an important molecule for the development of effective methods for the seafood sector. Following inspection missions from the European Union and United States of America, it was introduced in the Brazilian Residues Control Program to fulfill export and internal national requirements with a Maximum Residue Limit of 800?mu g/kg. A high performance liquid chromatography method with ultraviolet detection at a wavelength of 230?nm (??=?230?nm) for the detection of florfenicol in fish muscle was developed and validated according to the Brazilian Regulation 24/2009 (equivalent to European Union Decision 2002/657/EC). Fish samples were extracted with ethyl acetate and hexane followed by C18 solid phase clean-up and chromatographic separation on a reversed-phase C18 LC column with acetonitrile:water as mobile phase. The method results were also was compared with those obtained using liquid chromatography-tandem quadrupole mass spectrometry. The method meets the Brazilian regulatory requirements with a decision limit (CCa) of 840?mu g/kg and detection capability (CC beta) of 879?mu g/kg. This method is easy to use and has been implemented into Brazil's residue control program, with liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmation of any suspect samples using the same method. (c) 2012 John Wiley & Sons, Ltd.41125129Food and Agriculture Organization (FAO)International Atomic Energy Agency (IAEA) Joint Division of Nuclear Techniques in Food and Agriculture [D5-20-36]International Atomic Energy Agency (IAEA) Joint Division of Nuclear Techniques in Food and Agriculture [D5-20-36]