There has been increased activity in the last few years in the search for disease markers using fractionation of complex biological fluids combined with MS. While electrophoretic and chromatographic separations have played a major role in this endeavor, this manuscript is limited to a review of electrophoretic methods that have been established for disease biomarker discovery. These methods include 2-DE, difference gel electrophoresis (DIGE), and CE. We define what constitutes a biomarker, identify the steps required for establishing a biomarker, and describe the parameters needed in the design of an ideal diagnostic test. The application, advantages, and limitations of CE, DIGE, and 2-DE in meeting the goal of discovering novel biomarkers is discussed in detail, along with a few selected examples that illustrate the search for biomarkers for cancer and neurological diseases.