All drugs used in aquaculture must be approved by the government agency responsible for veterinary medicine; prudent use of antibiotics under veterinary supervision is critical in ensuring safety of aquaculture products. The international production of farmed turtle, considered a cultured aquatic species, has been growing continuously but control of bacteria proliferation in turtle farms requires frequent use of antimicrobials. In the present investigation, the residue depletion and withdrawal period of quinolone antibacterial oxolinic acid (OXO) was evaluated in Chinese softshell turtle Pelodiscus sinensis, after repeated (once daily for 5 days) oral doses of medicated feed containing OXO, at the dose of 30 and 60 mg/kg body weight (bw). Muscle and liver tissues were collected at 1, 2, 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, and 48 days posttreatment, and OXO was quantified using a validated high-performance liquid chromatography with diode array detector (HPLC-DAD) method. According to the test preparation record, the limit of detection (LOD) and limit of quantification (LOQ) for OXO were estimated at 0.03 and 0.1 µg/g, respectively. Considering a maximum residue level (MRL) of 0.1 µg/g for OXO in muscle in European Union (EU) legislative framework, the longest withdrawal period of 72 days was calculated on the basis of a safety span (corresponding to 50% of the time point when at which residues fall below the MRL) added to the depletion time (48 days). These results may be helpful not only for the establishment of public policies regarding the use of OXO for turtle farming but also to producers for proper handling to ensure safe consumption