Proteomics technologies demonstrate enormous data-gathering capabilities to discover disease-specific biomarkers in serum, plasma, urine, tissue and other biological samples. The traditional way to achieve this consists of protein separation performed using two-dimensional polyacrylamide gel electrophoresis (2DE). However, the 2DE approach has its drawbacks with respect to automation, sensitivity, and throughput. Considerable efforts have been devoted to the development of non-gel-based proteome separation technologies able to resolve complex protein and peptide mixtures prior to mass spectrometric (MS) analysis. This review discusses some of the most recent advances in multidimensional peptide separation techniques compatible with MS analysis, including gel electrophoresis, isoelectric focusing, capillary electrophoresis and liquid chromatography techniques. Based on future perspectives and our experiences, special attention is given to the application of two-dimensional chromatographic separation coupled to MS, the so-called multidimensional protein identification technology approach.