Abstract: The simultaneous analysis of all proteins expressed by a cell, tissue or organism in a specific physiological condition is the main goal of proteomic studies. Gel-based proteomic is the most popular and versatile method of global protein separation and quantification. This is a mature approach to screen the protein expression at the large scale. Based on two independent biochemical characteristics of proteins, two-dimensional electrophoresis combines isoelectric focusing, which separates proteins according to their isoelectric point, and SDS-PAGE, which separates them further according to their molecular mass. The next typical steps of the flow of gel-based proteomics are spots visualization and evaluation, expression analysis and finally protein identification by mass spectrometry. At present, two-dimensional electrophoresis allows simultaneously to detect and quantify up to thousand protein spots in the same gel in a wide range of biological systems for the study of differentially expressed proteins. However, gel-based proteomic has a number of inherent drawbacks. In this review article, the benefits, difficulties, limits and perspectives of gel-based proteomic approaches are discussed.