Abstract: A high performance liquid chromatography tandem mass spectrometric method was established for the determination of glucose concentration in serum with the aid of D-¹³C₆glucose internal standard. D-¹³C₆glucose was added to the serum sample in balance at room temperature. Then the protein was precipitated and extracted in anhydrous ethanol with centrifugation at low temperature. The supernatant was filtered through a 0.22 μm syringe filter into HPLC vials. Identification was achieved by electro-spray ionization (ESI) in positive mode using SIM mode. The calibration curve of glucose showed good linearity with correlation coefficient of R²=0.999 5, recoveries of spiked samples were in the range 100.1% to 102.8%, the detection limit of the method was 8 μg/kg (S/N=3), and the relative standard derivations were 0.49%. The established method is simple, accurate and precise, is appropriate for the determination of serum glucose with great accuracy.