The study was intended to evaluate the depletion of chloramphenicol (CAP) in rainbow trout (about 300-550 g body weight), after 10 days treatment with fish feedstuff containing chloramphenicol. A total of 60 animals were separated in two groups: one was fed with CAP containing feedstuff in order to have a dosage of about 80 mgkg(-1)day(-1), while a second group of fishes was fed with feedstuff not containing any CAP formulation (negative controls). The treatment was maintained for 10 days. After this period, groups of 2-5 animals were sacrificed at different withdrawal times up to a maximum of 31 days. Muscle tissues of each group of animals were then analysed for quantitative residual CAP determination both by enzyme linked immunoassay (ELISA) and liquid chromatography coupled to mass spectrometry (HPLC/MSMS). The methods applied were in house validated according to the guidelines laid down by the European Decision 657/2002/EC. Results and considerations are presented.