Fusion proteins constitute a class of engineered therapeutics and have emerged as promising candidates for disease treatment. However, the structural complexity and heterogeneity of fusion proteins make their characterization extremely challenging, and thus, an innovative and comprehensive analytical toolbox is needed. Here, for the first time, we demonstrate a novel and robust workflow to evaluate charge variants for a highly glycosylated fusion protein with heavy sialylation using imaged capillary isoelectric focusing (icIEF). In the development of the icIEF method, key factors that were systematically investigated include the desialylation level, the stability of the desialylated molecule, incubation time and temperature of desialylation, protein concentrations, urea and l-arginine effects on the tertiary structure, and instrumental comparability. Multivariate and correlation analyses were subsequently applied to confirm the impacts of the parameters evaluated. Furthermore, a microfluidic chip-based icIEF system coupled with ultraviolet detection and mass spectrometry (icIEF-UV/MS) was utilized to identify critical post-translational modifications and ameliorate the understanding of charge variants. Our study demonstrates that this workflow enables a mechanistic understanding of charge variants for heavily sialylated therapeutics.