Abstract: OBJECTIVE To establish an LC-MS/MS method for the determination of amphotericin B in plasma of Beagle dogs, and to study the pharmacokinetic parameters of liposomal amphotericin B(L-AmB) and reference drug(AmBisome) in Beagle dogs.METHODS Twelve adult healthy Beagle dogs, half male and half female, were selected. The design of double cross experiment was adopted, and the period of washout was 2 weeks, 6 mg·kg^-1(1.0 mg·mL^-1) was injected intravenously. The whole blood was collected before and different timepoints after administration. The concentration of amphotericin B in the plasma was determined by the validated LC-MS/MS method, and the pharmacokinetic parameters were calculated by DAS 2.1.1.RESULTS The main pharmacokinetic parameters of L-AmB and AmBisome were as follows: AUC_(0-t) was(705 520±178 252), (677 310±166 326) μg·h·L^-1; C_max was(142 683±29 823), (121 992±37 983)ng·mL^-1; t_1/2 was(8.9±1.5), (9.7±0.6)h.CONCLUSION There is no significant difference between the Beagle dogs after intravenous injection of L-AmB and the reference drug (AmBisome).