Donepezil is a medication used to treat dementia of the Alzheimer’s type. In this study, we developed and validated a quantitative analysis method for the determination of Donepezil in human plasma using liquid chromatography–tandem mass spectrometry(LC-MS/MS). As the internal standard, isotope-labeled material was used. After a simple protein precipitation using acetonitrile, the analytes were separated using 10 mM ammonium acetate (pH 4) and acetonitrile as mobile phase (isocratic condition) on a reversed-phase C18 column and analyzed by mass spectrometry in the multiple reaction monitoring (MRM) in positive scan mode. The method was linear in the concentration range of 0.5-50 ng/mL for donepezil (r > 0.999). The method was successfully verified for quantitative analysis of donepezil in human plasma.