Different methods for plasma peptidome preparation in peptidomics analysis had been compared in this study. Plasma proteins were either precipitated with acetonitrile or subjected to themal denaturation. The eluent was subjected to analyze by gel filtration high pressure liquid chromatography. The results indicated that acetonitrile could effectively precipitated plasma proteins, and had the best extracting effect. 2.5 fold acetonitrile was optimal. The results showed that the method was satisfactory and would be applied in peptidomics analysis of preparation.