Objective To establish the measurement method of DB02 in rat plasma by HPLC-MS/MS， operating the pharmacokinetic studies of DB02 in rat after tail vein injection. Methods Six Sprague Dawley（SD） rats divided into male and female were administrated by intravenous injection of a solution DB02 1.004mg/kg·bw， rat retinal venous plexus bloods were collected at different time points， the drug concentrations of plasma were determined by HPLC-MS/MS， calculating the main pharmacokinetic parameters by DAS2.0. Results The method exhibited a linear range of 4.60~930 ng/mL for DB02 in rat plasma， the lowest limit of quantification（LLOQ） was 4.60 ng/mL（S/N≥10）， to study recoveries， precision and accuracy of DB02 QC for 13.6， 123， 923 ng/mL， each concentration’s recoveries were above 90%， intra-day and inter-day precision were less than 15%， accuracy was 93.34%~102.51%， to meet the chemical drugs non-clinical pharmacokinetic study technical guidelines’ requirements. Pharmaco-kinetic parameters of DB02 in rats were as follows： C max=（513±69）μg/L； t 1/2=（33.4±5.3）min； AUC（0-t）=（8. 15±0. 06）mg/L·min. Conclusions HPLC-MS/MS was simple， rapid， accurate， and able to meet the pharmacokinetic repuirements of DB02 in rats. After 1.004mg/kg single intravenous administration of one single dose， distribution of DB02 in rats showed one compartment model.