The physiological activity of a significant subset of cell proteins is modified by the redox state of regulatory thiols. The cellular redox homeostasis depends on the balance between oxidation of thiols through oxygen and reactive oxygen species and reduction by thiol-disulfide transfer reactions. Novel and improved methodology has been designed during recent years to address the level of thiol/disulfide regulation on a genomewide scale. The approaches are either based on gel electrophoresis or on chromatographic techniques coupled to high end mass spectrometry. The review addresses diagonal 2D-SDSPAGE, targeted identification of specific redox-interactions, affinity chromatography with thioredoxins and glutaredoxins, gel-based and non-gel based labelling techniques with fluorophores (such as Cy3, Cy5, ICy), radioisotopes, or with isotopecoded affinity tags (ICAT), differential gel electrophoresis (DIGE) and combined fractional diagonal chromatography (COFIRADIC). The extended methodological repertoire promises fast and new insight into the intricate regulation network of the redox proteome of animals, bacteria, and plants.