Abstract: This study was designed to use iTRAQ technology coupled with 2D LC-MS/MS to study the comparative proteomics of different processing technology for pilose antler. 1015 proteins were identified with 2D LC combined with MOLDI TOF/TOF mass spectrometry. Comparative analysis with Protein Pilot(Version 4.5) revealed that 87 proteins were changed(P ≤ 0.05, the ratio of > 1.50 or < 0.60 as the threshold selection of difference proteins), of which 24 were up regulated and 33 were down regulated in the traditional frying process(TFP) compared with the fresh pilose antler(P ≤ 0.05). 7 significant different proteins(P ≤ 0.001), most of these significantly changed proteins were found to be involved in calcium ion binding and ATP binding associated with human healthy. Freeze drying with protective agent(FDP)(Trehalose) can improve the content of significantly different proteins(P ≤ 0.001) including Collagen alpha-1(XII) chain(COL12A1) and Collagen alpha-1(II) chain(COL2A1). The significant function involves in platelets activating, maintenance of spermatogonium, and disorder expression in tumor cells. The functional annotation by Hierarchical clustering and GO(gene ontology) showed that the main molecule functions of the proteins significantly changed in these processes were involved in binding(52.7%), catalytic(25.3%), structural molecule and transporter(6.6%).