A new technique for polymer microchannel surface modification, called in-channel atom-transfer radical polymerization, has been developed and applied in the surface derivatization of thermoset polyester (TPE) microdevices with poly(ethylene glycol) (PEG). X-ray photoelectron spectroscopy, electroosmotic flow (EOF), and contact angle measurements indicate that PEG has been grafted on the TPE surface. Moreover, PEG-modified microchannels have much lower and more pH-stable EOF, more hydrophilic surfaces and reduced nonspecific protein adsorption. Capillary electrophoresis separation of amino acid and peptide mixtures in these PEG-modified TPE microchips had good reproducibility. Phosducin-like protein and phosphorylated. phosducin-like protein were also separated to measure the phosphorylation efficiency. Our results indicate that PEG-grafted TPE microchips have broad potential application in bioniolecular analysis.