A sensitive and simple method is described for the determination of fluoroquinolones by high-performance liquid chromatography (HPLC) using a C18 column and fluorescence detection. The mobile phase was acetonitrile and 0.025M phosphate acid with 0.0025M sodium 1-heptanesulfonate monohydrate in water with a gradient program. The chromatographic conditions were optimized for the determination of ciprofloxacin, enrofloxacin, sarafloxacin, and flumequine in animal feed. The samples were extracted by a hydrochloric acid and acetonitrile followed by dilution in 0.01M oxalic acid at pH 4.0 and purified by solid-phase extraction. The procedure was validated by fortifying feed at 200, 1000, and 2000 µg/kg. The limits of detection and quantification were from 28.5 to 74.7 and 31.7 to 94.6 µg/kg, respectively. The average recoveries for fluoroquinolones were from 89.7 to 100.3%. The method was validated and shown to be efficient and precise for quantification of fluoroquinolones in animal feed. The results demonstrate the feasibility of the method for routine use to monitor these substances in feed.