A LC-MS method was established for the determination of the protein binding rates of oleanolic acid in human plasma and serum albumin.  The equilibrium dialysis combined with LC-MS to determine the total concentration in plasma and free drug concentration of oleanolic acid was carried out.  The human plasma protein binding rates of oleanolic acid at three concentrations were 79.6%, 81.9% and 63.3%, respectively.  The human serum albumin protein binding rates of oleanolic acid at three concentrations were 53.5%, 56.6% and 47.7%, respectively.  The method is shown to be simple, accurate, sensitive and specific for the determination of biological samples.  The protein binding rates in human plasma and serum albumin were of high strength.