Aim To determine in vitro the rat plasma protein binding rate by using microdialysis method.Methods The binding rate was determined by using microdialysis probe as sampling tools and zero-net flux method as calibrating method.The regression equation was made by the difference of concentrations between the dialysis sample and the perfusate.The(x-intercept) of regression equation was the free drug concentration(Cf).The plasma protein binding rate was calculated by using the following equation: f=(C0-Cf)/C0.Result The binding rate was kept relatively stable in the studied concentration range.Conclusion It is feasible that the plasma protein binding rate can be determined by using microdialysis method.