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Anti-microbials have been used to control the quality of the aquatic environment for both prophylactic and therapeutic purposes. Tetracyclines are among the main antimicrobials used in aquaculture, and present a particular difficulty for extraction, due to a complex structure and high interaction with components of the biological matrix. In this study, different techniques of extraction and clean-up of antimicrobials of the tetracycline class in tilapia filets have been optimized and compared, followed by validation of the methodology using the best procedure. Oxytetracycline, doxycycline, tetracycline and chlortetracycline were analyzed by HPLC-fluorescence under the following conditions: organic mobile phase composed of methanol:acetonitrile (1:1, v/v) and aqueous mobile phase containing sodium acetate (0.0375 mol L-1), calcium chloride (0.0175 mol L-1) and EDTA (0.0125 mol L-1) at pH 7.00. The chromatographic analysis was performed using a mobile phase gradient with a flow rate of 1 mL min(-1) and, detection wavelength of 385/528 nm (lambda(exc)/lambda(em)) Four extraction methods have been evaluated, namely: liquid-liquid partition; solid phase extraction (SPE) using phenyl, C18 and polymeric Oasis-HLB stationary phases; dispersive SPE (dSPE) using polymeric adsorbent XAD 16 resin; and QuEChERS. The methods have been optimized with fractional factorial experimental design and compared by the extraction efficiency. The liquid-liquid extraction and the QuEChERS methods showed low extraction efficiencies (14-30%) for the analytes. The use of dSPE showed good efficiency (40-60%), but with low precision and high consumption of time. Among the evaluated extraction techniques the use of SPE showed the best results, with emphasis on the phenyl phase (58-76%), and has been validated for analysis of residues of tetracyclines in tilapia muscle regarding selectivity, linearity, precision and limits of detection and quantification. The validated method was adequate for the investigation of the analytes at residue levels. (C) 2013 Elsevier B.V. All rights reserved.