Abstract: A quantitative method was developed for the simultaneous determination of five fluoroquinolones (FQs), including ciprofloxacin, enrofloxacin, norfloxacin, ofloxacin and pefloxacin, using ultra performance liquid chromatography-quadruple time of flight mass spectrometry (UPLC-QTOF). Samples were separated on Waters ACUITY UPLC BEH C18 column with gradient elution programs, using methanol and water containing 0.1% formic acid as the mobile phases, and analyzed in electrospray positive ion source, using three acquisition modes (MS, MS^E and MRM). Five FQs were quantified with an internal standard method. Limits of detection (LOD) and limits of quantification (LOQ) of the 5 FQs on the UPLC-QTOF were 0.04~0.22 μg/L and 0.17~0.90 μg/L, respectively. Linear ranges of the 5 FQs were 1~100 μg/L with good correlation coefficients (R²≥0.99). This method was compared with the UPLC-tandem mass spectrometry (UPLC-TQMS) method and showed comparable performance. UPLC-QTOF is fast, sensitive, and suitable for the simultaneous quantification of the 5 FQs in environmental water samples.