Abstract: Objective To develop and validate a normal phase HPLC-MS/MS method for the determination of donepezil enantiomer in human plasma. Methods Donepezil was extracted from plasma by n-hexane:isopropanol (98:2,V/V) with lidocaine serving as an internal standard. The analytes went through the column of CHIRALCEL OJ-H (250 mm×4.6 mm, 5 μm) with mobile phase n-hexane:n-propanol:diethylamine (60:40:0.1,V/V/V). Donepezil enantiomer was determined by API 3000 in MRM mode. Results The retention time of S-DN and R-DN were 15.56 min and 18.41 min, respectively. The calibration curves were linear in a range from 0.051 to 7.596 ng/mL for S-DN, and from 0.049 to 7.404 ng/mL for R-DN, respectively, both with more than 0.99 correlation coefficients. The relative recovery were 95.10%-103.70% for S-DN and 93.58%-98.00% for R-DN, respectively; the pretreatment recovery were 58.42%-61.08% for S-DN and 53.24%-61.87% for R-DN, respectively;; the within-day RSD ranged from 8.35% to 11.28% for S-DN and from 6.78% to 11.58% for R-DN, respectively; the between-day RSD ranged from 5.82% to 9.02% for S-DN and from 6.87% to 9.19% for R-DN, respectively. Conclusion This normal phase HPLC-MS/MS method is simple, rapid, sensitive and accurate for the determination of donepezil enantiomer in human plasma and is suitable for pharmacokinetic studies of donepezil enantiomers.