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CpG oligodeoxynucleotide 107 (CpG ODN107) is a novel radiosensitizer for glioma.Herein,a novel and sensitive reversed phase HPLC coupled with electrospray triple quadrupole mass spectrometry (LC-MS/MS) method followed one-step C18 solid-phase extraction (SPE) for biological matrix removal,had been developed and fully validated for the determination of CpG ODN107 and its metabolites such as 5’N-1,3’N-1,3’N-2 and 3’N-3 in mice plasma.The analytes were separated on an Extend-C18 analytical column (3.5 μm,150 mm × 2.1 mm) using an eluent of acetonitrile - 0.05％ NH3 aqueous (20:80,v/v) and detected by electrospray ionization (ESI) mass spectrometry in the negative multiple reaction monitoring mode (MRM).The assay was specific,and its linearity was superb (r > 0.998) for CpG ODN107 and its metabolites in the biological matrices.The precision,accuracy and relative recovery values were found to be < 15％,± 15％,and 95 - 105％,respectively.This method had been successfully applied to measuring concentrations of CpG ODN107 and its metabolites in mice plasma following intravenous administration of 15.0 mg/kg of CpG ODN107 in mice,which was suitable for the preclinical pharmacokinetic studies on CpG ODN107 and its metabolites.