Abstract: This study aims to develop a liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of ivabradine and N-demethylivabradine in human plasma, and investigate effects of stable isotope labeled (SIL) internal standard (IS) on ivabradine. The analytes and IS were extracted from plasma by protein precipitation with acetonitrile, and chromatographied on a Capcell PAK C₁₈ (100 mm × 4.6 mm, 5 μm) column using a mobile phase of methanol and 5 mmol·L^-1 ammonium acetate. Multiple reaction monitoring with electrospray ionization (ESI) was used in the positive mode for mass spectrometric detection. The effect of ivabradine isotope peak M+H+3⁺ on IS and the effect of SIL IS purity on ivabradine were evaluated. An appropriate concentration of SIL IS was chosen to permit method selectivity and linearity of the assay over the required range. The standard curves were demonstrated to be linear in the range of 0.100 to 60.0 ng·mL^-1 for ivabradine, and 0.050 0 to 20.0 ng·mL^-1 for N-demethylivabradine. The intra and inter day precision and accuracy were within the acceptable limits for all concentrations. Besides, the interaction between IS and ivabradine did not impact the determination of analytes. This method was successfully applied to a pharmacokinetic study of hydrogen sulfate ivabradine sustained release tablets on Chinese healthy volunteers.