The on-column reaction of glutathione with the reactive disulphide 2,2-dipyridyldisulphide was used for the specific detection of glutathione during micellar electrokinetic chromatography: In this set-up the front end of the capillary is used as the reaction chamber. 2,2-dipyridyldisulphide and a sample of glutathione are injected consecutively in the capillary as two discrete plugs separated with short plug of background electrolyte. Due to the differences in the mobilities of the 2,2-dipyridyldisulphide and glutathione on column mixing and reaction occur. This reaction is rapid and quantitative, because thiol form is stabilised by thiol-thione tautomerism. This fact also causes a large shift of the absorption maxima toward longer wavelength. Glutathione is thus quantitatively transformed into a mixed disulphide concomitantly with formation of equimolar amounts of the 2-thiopyridone that is further separated by micellar electrokinetic chromatography and determined spectrophotometrically at 343 nm.