A novel approach enables imaged capillary isoelectric focusing analysis of PEGylated proteinsPEGylation has been used as a strategy to enhance pharmacokinetic properties of therapeutic proteins by pharmaceutical industry. Imaged CIEF (iCIEF) is the current industry standard technology for pI determination and charge variant quantification of proteins and antibodies. However, the charge variants of PEGylated proteins merge into one broad peak during iCIEF, most likely due to masking of proteins by the surrounding PEG chain as well as the increased hydrodynamic volume due to PEGylation. Here, we report our novel matrix formula with a combination of glycine and taurine that significantly improved the separation of charge variants in PEGylated proteins. As a result, it is no longer necessary to conduct IEF of proteins prior to PEGylation, which does not reflect the changes caused by PEGylation and purification processes. The novel matrix (glycine and taurine) enables iCIEF analysis of PEGylated proteins in their real conjugated states.Note that 0.5% methyl cellulose solution, 1% methyl cellulose solution, iCE electrolyte kit, pI marker 4.65, pI marker 6.61, iCE3 Instrument with Alcott 720NV auto-sampler, and FC cartridge were purchased from ProteinSimple; the FC cartridge has a 50 mm, 100 μm (id) fluorocarbon-coated capillary Color online: See article online to view Figs. 1-7 in color.