After the first introduction of the concept of "proteome" more than 10 years ago, large-scale studies of protein expression, localization, activities and interactions have gained an exponential increase of interest, leading to extensive research and technology development. Proteomics is expansively applied in many areas, ranging from basic research, various disease and malignant tumors diagnostic and biomarker discovery to therapeutic applications. Several proteomics approaches have been developed for protein separation and identification, and for the characterization of protein function and structure. Two-dimensional gel electrophoresis, chromatography, capillary electrophoresis and mass spectrometry have become the most used proteomics methods. These techniques are also under constant development. This review provides an overview of the main techniques and their combinations, used in proteomics. The emphasis is made on description of advantages and disadvantages of each technique, to navigate in selection of the best application for solving a specific problem.