In contrast to the traditional biochemical study of single proteins or isolated pathways in health and disease, technical advances in the high-throughput screening of peptides by mass spectrometry have established new ways of identifying entire cellular protein populations in one swift analytical approach. This review discusses the recent progress in the biochemical analysis of skeletal muscle extracts and outlines the mass spectrometry-based proteomics approach for studying muscle tissues in normal and pathobiochemical processes using peptide mass fingerprinting. Individual topics covered include the most commonly inherited muscle disease, X-linked muscular dystrophy, the physiological process of fast-to-slow fibre transformation, and the role of fibre degeneration in age-related muscle wasting. Recent proteomic profiling studies of dystrophic muscles have revealed new disease markers in dystrophin-deficient fibres, such as adenylate kinase, the Ca2+-binding protein regucalcin and the small heat shock protein cvHSP. Since these muscle proteins are of low abundance, they have not previously been identified as biomarkers of muscular dystrophy, illustrating the increased sensitivity of modern mass spectrometric techniques. This review outlines comparative proteomic techniques that employ conventional labeling methods, such as Coomassie- or silver-staining. In addition, the most advanced proteomic screening approach currently available, fluorescence difference in-gel electrophoresis, is described and its potential for studying muscle proteomes is critically examined. As an alternative suggestion, the two-dimensional analysis of different protein samples separated in parallel on a single second dimension gel is introduced and the usefulness of this technique for direct comparative investigations is explained. The potential of studying protein complex formation by intraproteomics, estimating the composition of subcellular fraction by subproteomics, and analyzing total muscle protein extracts by mass spectrometry-based proteomics, is enormous. Proteomics is one of the most promising new analytical ways of comparing large muscle protein complements and has the potential to decisively improve modern biochemical and biomedical research into neuromuscular disorders.