Jugular vein (JV) blood sampling is widely employed in pharmacological, toxicological, and pharmacokinetic studies due to its ability to provide large and repeatable blood volumes. However, current approaches are constrained by low convenience, safety concerns, and variable sample quality. Here, we report the development of a new single-operator JV blood sampling technique for conscious rats. By optimizing animal restraint, puncture site localization, and procedural standardization, the method significantly reduced sampling time, puncture attempts, pain-related behaviors, and hemorrhagic injury, while ensuring consistent blood yield. Compared with the conventional approach, it achieved a 100% success rate, halved the average sampling time, and maintained stable serum biochemical, coagulation, and hematologic parameters. The utility of this method was further confirmed in an intraperitoneal glucose tolerance test and a cisplatin-induced acute kidney injury model, where data consistency was preserved. This technique provides a convenient, safe, and reliable alternative for repeated blood collection in conscious rats, ensuring animal welfare and supporting the generation of high-quality data in preclinical research.
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