This paper investigated the potential use of silica nanoparticles (SNPs) as pseudostationary phase (PSP) for protein separation. The wall adsorption of SNPs as well as the influences of SNPs and poly(ethylene oxide) (PEO) were studied. The SNPs showed selectivity toward the proteins, and the concentration ratio of SNPs to PEO influenced the separation. The proteins that could not be baseline-resolved under conventional CZE mode were separated in a buffer consisting of 30 mM phosphoric acid, 0.05% PEO, and 0.05% SNPs at pH 2.37, with detection limits ranging from 2 to 45.5 ppm. The intraday and interday RSDs of the migration times were in the ranges of 2.1-2.8% (n = 5) and 2.5-3.4% (three days, n = 3 x 5 = 15), respectively.