Abstract: OBJECTIVE To identify the related substances in tegafur by hyphenated techniques for its manufacturing process and quality control. METHODS A column packed with the octadecyl silane chemically bonded silica was used for the separation of the related substances with a mixture of acetonitrile-methanol and 10 mmol·L^-1 ammonium acetate buffer solution as the mobile phase by isocratic elution. The structures of the related substances were speculated and identified by electrospray positive ionization LC-TOF/MS accurate ion mass and MS/MS determination and elucidation. RESULTS & CONCLUSION Tegafur and the related substances are separated under the established HPLC condition. Four related substances in tegafur are characterized by hyphenated techniques. Their structures are elucidated as the different substituents having the same frame structure as tegafur and starting material.