Objective To establish an HPLC-MS/MS method for determination of the plasma concentration of granisetron hydrochloride administered by transdermal patch in Beagle dogs and evaluate its pharmacokinetics.Methods Six beagle dogs were given topically applied transdermal patches (72 h) for 6 times with an interval of 24 h. HPLC-MS/MS was applied to determine the concentration of granisetron hydrochloride in blood samples, thereafter the pharmacokinetic parameters were calculated.Results The trough concentration of Cmin was 0.0459 ng/mL and 0.2593 ng/mL after the first-dosage and sixth-dosage of granisetron transdermal patches, respectively. The peak concentration of Cmax was 0.4637 ng/mL and 0.4637 ng/mL, respectively. The AUC_0~96 was 18.92 h·ng/mL and 40.21 h·ng/mL, respectively. The Tmax was 48.3 hours and 45.3 hours, respectively, and the MRT_0-96 was 48.9 hours and 45.84 hours, respectively.Conclusions The concentration of granisetron has accumulation effect after repeated administration of granisetron hydrochloride transdermal patches, and it reaches a steady state on the sixteenth day by multiple administrations.