This work presents improved protease digestion conditions for membrane protein detection. Two improved conditions, 0.01% SDS or the combination of 10% MeOH and 0.01% RapiGest, were chosen and applied to proteins from Escherichia coli. The application of both improved conditions to a membrane protein fraction of Escherichia coli resulted in the identification of 309 (SDS) and 329 (MeOH/RapiGest) unique proteins of which 140/309 and 148/329 were membrane proteins.