Acteoside(verbascoside), a phenylethanoid glycoside widely distributed in various plants, has been shown to have potential activity against Alzheimer’s disease, attracting great attentions recently. The present study was designed to develop a selective and sensitive LC-MS/MS method for the determination of acteoside in biological samples and carry our a phamacokinetic(PK) study in beagle dogs. The PK parameters were calculated using non-compartmental models. Following a single-dose oral administration, acteoside was rapidly absorbed and eliminated, with Tmax being between 30 to 45 min and terminal half-life being about 90 min. The areas under the time-concentration curve(AUC) were 47.28 ± 8.74, 87.86 ± 13.33, and 183.14 ± 28.69 mg·min·L-1 for oral administration of 10, 20, and 40 mg·kg-1, respectively, demonstrating that the exposure of acteoside proportionally increased with the dose level. The absolute bioavailability of acteoside was around 4%. For all the PK parameters, there were large variations between individual dogs. In conclusion, the pharmacokinetic characteristics observed in the present study can be of great value to help better understand the pharmacological properties of acteoside and to improve the outcome of its clinical use. Acteoside (verbascoside), a phenylethanoid glycoside widely distributed in various plants, has been shown to have potential activity against Alzheimer’s disease, attracting great attentions recently. The present study was designed to develop a selective and sensitive LC-MS / MS method for the determination of acteoside in biological samples and carry our a phamacokinetic (PK) study in beagle dogs. The PK parameters were calculated using non-compartmental models. Following a single-dose oral administration, acteoside was fast absorbed and eliminated, with Tmax being between 30 to 45 min and terminal half-life being about 90 min. The areas under the time-concentration curve (AUC) were 47.28 ± 8.74, 87.86 ± 13.33, and 183.14 ± 28.69 mg · min · L-1 for oral administration of 10, 20 , and 40 mg · kg -1, respectively, demonstrating that the exposure of acteoside proportionally increased with the dose level. The absolute bioavailability of acteoside was around 4%. For all the PK parameters, there In conclusion, the pharmacokinetic characteristics observed in the present study can be of great value to help better understand the pharmacological properties of acteoside and to improve the outcome of its clinical use.