Despite all remarkable progress in gel-based proteomics in recent years, there is still need to further improve quantification by decreasing the detection limits and increasing the dynamic range. These criteria are achieved best by fluorescent dyes that specifically stain the proteins either by adsorption after gel electrophoresis (in-gel staining) or covalent coupling prior to gel electrophoresis (in-solution staining). Here we report a multiplex analysis of protein samples using maleimide-activated cyanine-based (Cy3 and Cy5) and rhodamine-based dyes (Dy505, Dy535, and Dy635) to permanently label all thiol-groups of cysteine-containing proteins. The detection limits in SDS-PAGE were about 10 ng per band and even 2 ng for BSA due to its high content of cysteine residues. Thus only 5 microg protein of a mouse brain homogenate were analyzed by 2-DE. Both cyanine- and rhodamine-based dyes also stained proteins that did not contain cysteines, probably by reaction with amino groups. This side reactivity did not limit the method and might even extend its general use to proteins missing cysteine residues, but at a lower sensitivity. The dynamic range was more than two orders of magnitude in SDS-PAGE and the Dy-fluorophores did not alter the mobility of the tested proteins. Thus, a mixture of Dy505-, Dy555-, and Dy635-labeled Escherichia coli lysates were separated by 2-DE in a single gel and the three spot patterns relatively quantified.