Although the membrane cytoskeletal protein dystrophin of 427kDa and its tightly associated glycoprotein complex are drastically affected in muscular dystrophy, recent large-scale proteomic investigations did not identify full-length dystrophin in muscle preparations and were unable to determine its molecular fate in dystrophinopathy. Because conventional two-dimensional gel electrophoresis underrepresents many low-abundance and membrane-associated protein species and in-gel trypsination is often hampered by an inefficient digestion of certain target proteins, here we have applied direct on-membrane digestion of one-dimensional blots of the sarcolemma-enriched fraction and the isolated dystrophin-glycoprotein complex. This method succeeded in the mass spectrometric identification of dystrophin isoform Dp427 and associated glycoproteins as well as sarcolemmal dysferlin. In addition, protein bands representing established signature molecules of cross-contaminating membrane systems, such as the voltage-sensing dihydropyridine receptor of transverse tubules, the ryanodine receptor Ca2+-release channel of triad junctions, and the Ca2+-ATPase of the sarcoplasmic reticulum, were identified by mass spectrometry. Thus, proteomic approaches using on-membrane digestion might be suitable for future studies of low-abundance proteins, integral proteins, peripheral membrane proteins, and high-molecular-mass proteins. On-membrane digestion has the potential to develop into the method of choice for studying these classes of proteins, whose presence is otherwise missed by conventional gel electrophoresis-based proteomics.