Fluoroquinolones (FQs) are among the most important antibacterial agents (synthetic antibiotics) used in human and veterinary medicine. An analytical method based on reversed-phase liquid chromatography with fluorescence detection was developed and validated for the simultaneous determination of nine FQs and the quinolone pipemidic acid in urban wastewater. Aqueous samples were extracted using mixed-phase cation-exchange disk cartridges that were subsequently eluted by ammonia solution in methanol. Recoveries were above 80% at an overall precision of better than 10%. Instrumental quantification limits varied between 150 and 450 pg injected. The presented method was successfully applied to quantify FQs in effluents of urban wastewater treatment plants. The two most abundant human-use FQs, ciprofloxacin and norfloxacin, occurred in primary and tertiary waste-water effluents at concentrations between 249 and 405 ng/L and from 45 to 120 ng/L, respectively. The identity of FQs in urban wastewater was confirmed by recording full fluorescence spectra and liquid chromatography directly coupled to tandem mass spectrometry. These results indicate that conventional environmental risk assessment overestimates FQ concentrations in surface waters by 1 to 2 orders of magnitude.