An analytical method based on HPLC with MS/MS detection was developed and optimized in order to determine the most useful antibiotics (sulphonamides and tetracyclines) used in aquaculture. A simple extraction procedure, without any clean-up step, was evaluated in order to obtain maximum analyte recovery from fish samples ( Sparus aurata). A mixture of methanol:water 70:30 (v/v)+1 mL EDTA 0.1 M was selected as optimum extractant solution. Because no matrix effects were observed, a standard calibration curve prepared in mobile phase was used for quantification purposes. Antibiotic-free fish samples were spiked at different concentration levels and analysed by the optimized HPLC method. The average recoveries ( n=6) obtained were satisfactory, ranging from 88% to 110% at 100 μg kg−1. The proposed methodology provided limits of detection for the tested antibiotics in the 1.2–16 μg kg−1 range, lower than 100 μg kg−1, the maximum residue level established by the European Union. Finally, commercial fish samples from different origins were analysed in order to confirm the usefulness of the developed methodology. [ABSTRACT FROM AUTHOR]