Identification of proteins and characterization of posttranslational modifications are crucial steps for many biological, biochemical, and biomedical studies, and mass spectrometry has become the method of choice for these analyses. Here we describe two methods for the on-membrane digestion of proteins electroblotted onto nitrocellulose membranes prior to analysis by mass spectrometry. These on-membrane methods take approximately half the time of in-gel digestion and provide better digestion efficiency, due to the better accessibility of the protease to the proteins adsorbed onto the nitrocellulose, and better protein sequence coverage, especially for membrane proteins where large and hydrophobic peptides are commonly present.