objective:To establish optimized preparation method of rat osteoblasts secretome sample for two-dimension electroph-oresis(2-DE) and set a foundation for further study of osteoblasts secretome. Methods:By using dialysis, dialysis-trichloracetic acid(TCA)-acetone precipitation and dialysis-acetone precipitation methods respectively, the rat osteoblasts secretome samples were prepared, and then the most optimized preparation method was determined by comparing the 2-DE results. Results: Dialysis-acetone precipitation method was the most optimized method, which lost less proteins, increased its solubility, made 2-DE images more clearly and obtained high resolution. Conclusion:Dialysis-acetone precipitation method is the most optimized preparation method for rat osteoblasts secretome 2-DE sample.