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Objective To develop a HPLC-MS/MS method for detecting the isosorbide 5-mononitrate in human plasma.Method Theophylline was used as internal standard (IS).The plasma sample was prepared by solid phase extraction (SPE), and separated on Agilent ZORBAX Extend-C18(4.6×150 mm,5 μm)column with a mobile phase of acetonitrile-water (2.5mM ammonium acetate)30-70 (v/v).The flow rate was 1.0 mL/min and the column temperature was 25℃.A tandem mass spectrometer equipped with electrospray ionization (ESI) was used as detector and operated in the positive ion mode.Multi reaction monitoring (MRM) using the precursor → product ion combinations of m/z 249.8→ 58.9 and rn/z 178.8→ 163.8 were used to quantify isosorbide 5-mononitrate and the internal standard respectively.Results The calibration curve was linear in range of 10 ~ 1000 ng/mL (r<0.998), endogenous components could not interference the determination method, the coefficient variation (CV) of IS-normalized matrix factor (MF) was less than 7%, the recovery of interest and IS were stable and CV were less than 15%, the with-run and between-run CV of accuracy and precision were less than 11%.Conclusion: The method is rapid, sensitive, and reproducible.It is suitable for the determination of isosorbide 5-mononitrate in human plasma.