Doxorubicin (Dox) is a drug widely used in cancer chemotherapy. Dox is essential in the treatment of pediatric patients diagnosed with different types of cancer, including osteosarcoma (OS). Dox has been used clinically for more than 3 decades, but recently it has been recognized that the cytotoxic effect occurs by multiple mechanisms that have not yet been conclusively identified. Known risk factors for Dox include cumulative dose, younger age, concomitant use of some other antineoplastic agents, female gender, and increased dose intensity. For these reasons, it is important to develop and improve plasma quantification methods for this antineoplastic drug, with the idea that they are more easily usable in clinical practice and thus seek to improve the treatment of patients using Dox. The objective of the present study was to develop an economical, precise, rapid, and exact analytical method by HPLC for the determination of Dox for pharmacokinetic studies in pediatric patients. This method was successfully applied to study Dox chronopharmacokinetics in pediatric patients with osteosarcoma and thus evaluate its chronotoxicity.