A method was optimized and validated for simultaneous estimation of some antibiotics such as chlortetracycline (CTC), doxycycline (DOX), florfenicol (FF), flumequine (FLU), nalidixic acid (NAL), sulfadiazine (SDI), sulfathiazole (STZ) and trimethoprim (TMP) in fish muscle and water samples. The method is based on solid phase extraction (SPE) and simple extraction followed by high performance liquid chromatography with ultraviolet detection (HPLC-UV). The HPLC method was optimized using experimental design. The optimum conditions for separation determined with the aid of central composite design were: (1) initial mobile phase concentration: 0.1% formic acid in water/acetonitrile (90/10, v/v), (2) column temperature 25 °C and (3) mobile phase flow rate (1.2 ml min⁻¹). The optimized method was validated according to ICH guidelines. The detection and quantification limits were between 0.2–0.4 and 0.3–0.6 μg kg⁻¹, respectively, for fish and between 0.005–0.02 and 0.01–0.08 μg ml⁻¹, respectively, for water. The procedure was also applied to the analysis of spiked Nile tilapia samples. Three antibiotics (SDI, CTC and FF) were orally administered and the residue was analyzed using liquid chromatography-electro spray ionization-mass spectrometry with positive ion mode (LC-ESI/MS).