OBJECTIVE： To develop the method for the determination of amoxicillin concentration in dog plasma， and to study its pharmacokinetic characteristics. METHODS： Totally 12 Beagle dogs were collected and given Amoxicillin capsules 250 mg intragastrically. The 1.0-1.5 mL plasma samples were collected before medication， and 0.5， 1， 1.5， 2， 2.5， 3， 3.5， 4， 4.5， 5， 5.5， 6， 8， 10 h after medication. Using amoxicillin-d4 as internal standard （IS）， after precipitated with acidic acetonitrile， the plasma concentration of amoxicillin was determined by LC-MS/MS. The separation was performed on an Atlantis dC18 column with mobile phase consisted of acetonitrile （containing 0.1％ formic acid）-10 mmol/L ammonium formate water solution （pH 4.5， gradient elution） at the flow rate of 0.3 mL/min. The column temperature was 30 ℃， and sample size was 1 μL. Mass spectrum condition included ESI， positive ion mode， multiple reaction monitoring， ion source spray voltage 5.5 kV， ion source temperature 450 ℃， atomizing gas pressure 30 psi， heater pressure 40 psi， ion pair m/z 366.2→113.9 and m/z 370.2→353.0 for amoxicillin and IS， fragmentation voltage 26 and 16 V， residence time 200 ms. RESULTS： The linear range of amoxicillin was 0.1-16 μg/mL （r＝0.998 2）. RSDs of intra-day and inter-day were lower than 14.3％ and 11.8％ （n＝5）. The accuracy was 93.0％-112.0％ （n＝3）. The extraction recoveries were 58.9％-63.3％ （RSD＝3.3％， n＝3）， and matrix effect was 2.8％（n＝6）. RSDs of stability test was lower than 15％ （n＝3）. By single compartment model， the pharmacokinetic parameters of amoxicillin included that t1/2 was 1.56 h， AUC1-10 h was 24 300 ng·h/mL and cmax was 7 523 ng/mL. CONCLUSIONS： The method is sensitive， accurate， simple and rapid. It is applied for the determination of amoxicillin concentration in the plasma of dog and the pharmacokinetic study of amoxicillin.