An improved hydrophilic interaction liquid chromatography (HILIC) method has been developed to separate members of a closely related family of chemoprotective phytochemicals called glucosinolates. This method exploits the emergence of a second generation of HILIC chemistry, using a silica-based permanently zwitterionic stationary phase. These columns are more robust, durable, and glucosinolates separations are more reproducible than with the original polyhydroxyethyl aspartamide columns. Furthermore, the HILIC system that we report herein permits much greater alteration of the mobile phase composition for customized separation of glucosinolates from plant extracts, across a wide spectrum of polarity.