The iCE280 Analyzer (iCE280) was evaluated for its potential application as a high-throughput tool to determine pI and separate charge related species using glycosylated, non-glycosylated and pegylated protein therapeutics as models. Resolution was achieved for glycosylated and non-glycosylated molecules, but remained a challenge for pegylated proteins. The sources of charge variants were determined to be the presence of C-terminal lysine residues, sialic acid content, and deamidation. Limited assay performance evaluation demonstrated that the method was linear in the concentration range of 2–333 μg/ml of IgG with linear regression coefficients of 0.984, 0.998, and 0.990 for acidic, main and basic species, respectively. Limit of detection and limit of quantitation were determined to be 3 and 11 μg/ml. The R.S.D. for intra- and inter-day precision as well as reproducibility was determined to be 0.2% or less for all pI values and 1.4% or less for acidic and main peak area distribution; the R.S.D. for basic peak area distribution was 5.7% or less. Robustness testing was performed by deliberately deviating ±50% of pharmalyte concentration away from the desired condition. This deviation revealed a pI shift of only 0.06 units and resulted in no significant impact on area percent distribution. Utilization of iCE280 Analyzer eliminated the mobilization step associated with traditional capillary isoelectric focusing analysis and increased analytical throughput at least 2-fold.