OBJECTIVE：To establish the method for determining protein binding rate of dipeptidyl peptidase- 4 inhibitor LGT- 6 in different species of plasma ，and to compare their difference. METHODS ：By equilibrium dialysis ，LGT-6（3，30，300，3 000 nmol/L）was equilibrated in rat ，monkey and human plasma （i. e. internal dialysis solution ）for 48 h，using phosphate buffer as the external dialysis solution. The concentration of LGT- 6 in internal and external dialysis solution was determined by UPLC-MS/MS using tolbutamide as internal standard ，and the plasma protein binding rate was calculated. The determination was performed on ACQUITY UPLC HSS T 3 column with water （containing 0.01％ formic acid ）-acetonitrile（containing 0.01％ formic acid ）as mobile phase at the flow rate of 0.6 mL/min. The column temperature was 40 ℃，and the sample size was 2 μL. The ion source was electrospray ion source ，and the multiple ion monitoring mode was used to carry out positive ionization scanning. The ion pairs for quantitative analysis were m/z 487.0→434.3（LGT-6），m/z 271.1→172.0（internal standard ），respectively. RESULTS ：At the concentrations of 3，30，300，and 3 000 nmol/L，the protein binding rates of LGT- 6 in rat plasma were （96.25±0.97）％，（84.16± 1.24）％，（78.25±0.61）％，（66.63±0.95）％；the protein protein binding rates in monkey plasma were （98.54±0.58）％，（87.27± 1.01）％，（79.35±0.86）％，（66.69±0.54）％；the protein binding rates in human plasma were （99.40±1.03）％，（84.48± 1.15）％，（77.62±0.77）％，（66.93±0.48）％. At the same concentration ，the protein binding rates of LGT- 6 in rat ，monkey and human plasma had no significant difference （P＞0.05）. In the same species of plasma ，there were significant differences in the plasma protein binding rates of different concentration of LGT-6 among those groups （P＜0.05），and it decreased with 才〔2016〕4015） the increase of drug concentration. CONCLUSIONS ： The method for the determination of plasma protein binding rate of LGT-6 is successfully established. The data revealed that the protein binding rate of LGT- 6 is concentration-dependent ， there was no obvious spec ies difference on protein binding rates of LGT- 6 in rat ，monkey and human plasma under the same concentration.