Ultra-high performance liquid chromatography coupled to fluorescence detection has been proposed for the determination of thirteen quinolones of human and veterinary use (danofloxacin, sarafloxacin, difloxacin, flumequine, norfloxacin, pipemidic acid, enoxacin, lomefloxacin, marbofloxacin, ciprofloxacin, enrofloxacin, moxifloxacin and oxolinic acid). Sample treatment consisted of a modified method based on salting-out assisted liquid–liquid extraction, which involves the use of magnesium sulphate and sodium chloride as salting-out agents. To demonstrate the applicability of the method, it was characterized for three different matrices of interest (milk, urine and environmental water), obtaining very low limits of quantification (0.2–192 μg L−1). The precision of the method was evaluated in terms of repeatability and intermediate precision, and the results were acceptable in all cases (relative standard deviations lower than 11%). Recovery studies were performed on the three matrices, obtaining values between 71% and 104%.