Objective To study the protein binding rate of glaucocalyxin A in plasma of rat. Methods The plasma balance dialysis was used. An HPLC for the quantitative determination of glaucocalyxin A was presented and used to calculate the protein binding rate in plasma of rat. Results There were protein binding rate results of 74.46%, 77.87%, and 75.29% at three various concentrations 20, 10, and 1 ?g/ mL of glaucocalyxin A in plasma of rat. Conclusion The HPLC method used to determine glaucocalyxin A is simple, rapid, and sensitive with good specificity, precision and accuracy, and glaucocalyxin A has medium capacities in protein binding rate in plasma of rat.