Large part of the current research in biology, medicine, and biotechnology depends on the analysis of DNA (genomics), proteins (proteomics), or metabolites (metabolomics). The advances in biotechnology also command development of adequate analytical instrumentation capable to analyze minute amounts of samples. The analysis of the content of single cells may serve as an example of ultimate analytical applications. Most of the separation techniques have been developed in the last three decades and alternative approaches are being investigated. At present, the main protocols for analyses of complex mixtures include 2-DE (IEF) followed by electrophoresis in SDS polyacrylamide gel (SDS-PAGE) and chromatographic techniques. Information-rich techniques such as MS and NMR are essential for the identification and structure analysis of the analyzed compounds. High resolution separation of the individual sample components is often a prerequisite for success. High resolution proteomic analysis in the majority of laboratories still relies on the time consuming and laborious offline methods. This review highlights some of the important aspects of 2-D separations including microfluidics.