Cellular aging is a fundamental biological process, and mass spectrometry-based proteomics has been widely used for the global identification of age-related changes in a variety of tissues. The proteomic profiling of senescent skeletal muscles has revealed a variety of alterations in proteins associated with the contractile apparatus, cell signaling, ion homeostasis, metabolism, and the cellular stress response. Here, we outline the two-dimensional gel electrophoretic separation and fluorescent labeling of the urea-soluble protein complement from aged diaphragm muscle. This chapter describes the various experimental steps involved in gel electrophoresis-based proteomics, including protein extraction, isoelectric focusing, slab gel electrophoresis, fluorescence labeling, image analysis, protein digestion, mass spectrometric identification of proteins and immunoblotting.