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This book presents a depletion study of sulfadiazine and trimethoprim in muscle plus skin of gilthead sea bream (Sparus aurata). The fish were held in seawater at a temperature of 24-26 oC and fed with medicated feed for five consecutive days at daily doses of 25 mg of sulfadiazine and 5 mg of trimethoprim per kg of fish body weight. The premix trimethoprim-sulphadiazine 50 % was used for the preparation of the medicated feed. Two different diets, fish oil and plant oil -based diets were investigated. The method for the extraction of the analyte residues from fish tissue was developed and validated according to the requirements of the European Decision 2002/657/EC. Liquid chromatography-mass spectrometry was used for the determination of the analytes. Considering a Maximum Residue Limit (MRL) of 100 mig/kg for the total of sulfonamides and 50 mig/kg for trimethoprim residues in fish muscle plus skin, the withdrawal period of the premix trimethoprim-sulphadiazine 50 % was calculated in both diets. The investigation of this work is important in order to protect consumers by controlling the undesirable residues in fish.