A gel absorption-based sample preparation method for shotgun analysis of membrance protcome has been developed. In this new method, membrance proteins solbilized in a strating buffer containing a high con-centration of sodium dodecyl sulfate(SDS) were directly entrapped and immobilized into gel. After the detergent and other salts were removed by washing the proteins were subjected to in-gel digestion and the tryptic peptieds were extracted and analyzed by capilary liquid chromatorgraphy coupled with tandem mass spectrometry (Caplc-MS/MS). The results showed that the newly developed method not only avoided the protein loss and the adverse protein modifications during gel embedment but also only avoided the protein loss and the adverse protein modifications during gel embedment but also improved the subsequent in-gel digestion and the recoery of trypic pepides, particularly the hydropho-bic peptieds, thereby facilitating the identification of membrane proteins, especially the integral mem-brane proteins compard with the conventiona tube-ge; digestion method the newl developed method increased the mumbers of identified membrane proteins and integral membrane proteins by 25.0% and 30.2%, respectively demonstration that the method is of broa practicability in gel-based shot-gun analysis of membrane proteome.