The comprehensive analysis of gene expression in complex biological systems has demanded the development of new technologies to study the cell transcriptome and the cell proteome. Each approach has advantages and disadvantages from both the conceptual and the methodological viewpoints. Differential proteomics, the comparison of distinct proteomes (eg normal versus diseased cells, diseased versus treated cells etc) is of paramount importance. Several approaches can be used and these typically involve electrophoresis and/or chromatography combined with chemical or metabolic labelling and mass spectrometry. These approaches aim to identify molecular targets, namely proteins, involved in different physiopathological states. Incorporating this knowledge with knowledge from other technologies lays the foundations of active principles at the molecular level. Here, the various gel- and non-gel-based approaches that are used in a wide range of biological systems for the study of differentially expressed proteins will be reviewed.