A novel sorbent material of ultrapure silica gel provided with novel State of the Art Bonding- and Endcapping Technology commercially available under the name PerfectSil Target (250 x 4 mm, ODS-3, 5 microm, by MZ-Analysentechnik, Germany) was used and validated for the sensitive HPLC determination of ten quinolone antibiotics: enoxacin, ofloxacin, norfloxacin, ciprofloxacin, danofloxacin (DAN), enrofloxacin (ENR), sarafloxacin, oxolinic acid (OXO), nalidixic acid (NAL), and flumequine. The analytical column validation was performed in terms of separation efficiency, precision, and peak asymmetry. The separation was achieved at ambient temperature using a mobile phase of TFA (0.1%)-CH3OH-CH3CN delivered under the optimum gradient program, at a flow rate of 1.2 mU/min. Photodiode array detection was used and eluant was monitored at 275 nm. For the quantitative determination caffeine (7.5 ng/microL) was used as internal standard. The achieved LODs were 0.03 ng/microL per 50 microL injected volume for OXO, 0.1 ng/microL for DAN, ENR, and NAL, and 0.2 ng/microL for the remaining six studied quinolones. The method was validated in terms of interday (n = 6) and intraday (n = 5) precision and accuracy. The proposed method was successfully applied to the analysis of pharmaceutical formulations destined either for human or for veterinary use.