We have investigated the behavior of thiol-ene substrates that is a class of promising materials for lab-on-a-chip electrophoresis applications. Two polymeric materials were prepared by copolymerization of N,N-dimethylacrylamide (DMA), (3-(methacryloyl-oxy)propyl)trimethoxysilane (PMA) and 3-trimethylsilanyl-prop-2-yne methacrylate (MAPS) and specifically adapted to thiol-ene formulations in order to obtain a neutral and permanent coating for microchannels. The performance of two different thiol-ene substrates (with 20 % and 40 % excess of thiol groups, respectively) coated with either p-(DMA-PMA) or p-(DMA-PMA-MAPS) copolymer were evaluated in terms of surface hydrophilicity, suppression and stability of electro-osmotic flow and prevention of protein adsorption. Surface modification of thiol-ene containing a 20 % excess of thiols with the terpolymer p-(DMA-PMA-MAPS) was found to offer the most stable coating and most efficient charge shielding in the pH range from 3 to 9. The modified microchannels were successfully applied to electrokinetic separations of acidic and basic proteins. [Figure not available: see fulltext.]