A new stationary phase based on wide-pore hybrid silica bonded with amide ligand has been used toexplore the utility of HILIC for the analytical characterization of protein biopharmaceuticals. Various,highly-relevant samples were tested, including different insulins, interferon a-2b and trastuzumab. Thiswork shows that HILIC can be successfully employed for the analysis of therapeutic proteins and mAbs,using mobile phase compositions comprised of between 65 and 80% ACN and 0.1% TFA. In terms of elutionorder and selectivity, these HILIC separations have proven to be highly orthogonal to RPLC, while thekinetic performance remains comparable. In the case of characterizing trastuzumab, HILIC was uniquelyable to resolve several important glycoforms at the middle-up level of analysis (fragments of 25–100 kDa).Such a separation of glycoforms has been elusive by other separation mechanisms, such as RPLC and IEX.Besides showing orthogonality to RPLC and improved separations of glycoforms, HILIC offers severaladditional benefits for biopharmaceutical characterization: i) an inherent compatibility with MS, ii) areduced requirement for very high mobile phase temperatures that are otherwise needed in RPLC tolimit undesirably strong adsorption to the surface of the stationary phase, and iii) the possibility tocouple several columns in series to improve resolving power, thanks to comparatively low mobile phase viscosity.