To establish kinetic assay method for the analysis of hemolysis and to investigate dynamic  hemolytic process of polysorbate 80.  The UV-VIS spectrum of heme changes when hemoglobin is released   continuously during the hemolytic process.  Therefore, dynamic hemolytic curve was determined as a new way to characterize the kinetic process of interaction between polysorbate 80 and red blood cells.  The effect of  polysorbate 80 on blood cells could be perfectly investigated by the hemolytic dynamics.  Dynamic hemolytic parameters of polysorbate 80 were calculated according to the hemolytic curves.  The constants of hemolytic rate and maximum hemolytic rate of polysorbate 80 had fine linear relationships at the range of 1−20 mg·mL⁻¹ and 5−20 mg·mL⁻¹, respectively.  In comparison with the present official method such as macroscopic observation and static spectrophotometric methods, kinetic spectrophotometry has the advantages of real time, on-line    determination, sensitive, objective, good reproducibility and 2-dimensional information acquired.  Therefore, as a biological technique, kinetic spectrophotometry could be applied to evaluate the quality of polysorbate 80 and to screen other solubilizing excipients.