Monodisperse-porous microparticles functionalized with glutathione by thiol-ene chemistry were synthesized as a new stationary phase for hydrophilic interaction chromatography. Monodisperse-porous poly(3-trimethoxysilylpropyl methacrylate-co-ethylene dimethacrylate), poly(TMSPM-co-EDMA) microspheres (6 μm in size) were obtained with different seed latex/monomer ratios and diluent compositions by multistage microsuspension copolymerization. The zwitterionic chromatographic ligand containing thiol moiety (i.e. glutathione) were covalently attached onto the TMSPM attached-poly(TMSPM-co-EDMA) microparticles. The selected starting material allowed the direct attachment of zwitterionic ligand onto the support material using thiolene chemistry. The derivatized microparticles were slurry packed into the microbore columns with 2 mm i.d. and used as stationary phase for the separation of nucleosides in hydrophilic interaction chromatography with the plate numbers up to 54.000 plates/m. The results showed the usability of tailored poly (TMSPM-co-EDMA) microparticles as a stationary phase and thiolene chemistry in the manufacture of a chromatographic stationary phase with high efficiency in hydrophilic liquid chromatography applications.