Glycosylation is a key determinant of the efficacy, stability, and pharmacological behavior of therapeutic proteins. The presence of two N-glycosylation sites (Asn25 and Asn80) on R27T, an engineered variant of interferon-β1a, increases its structural complexity and analytical demands. In this study, we established an integrated workflow for comprehensive glycan profiling of R27T by refining existing analytical tools. For total glycan analysis, optimized fluorescent labeling was performed using procainamide, which provided enhanced sensitivity and broader glycan coverage than conventional 2-aminobenzamide. To evaluate the reliability of site-specific glycan quantification, liquid chromatography-tandem mass spectrometry-based peptide mapping results were systematically compared with fluorescence-based profiling, a widely accepted quantitative standard. The correlation varied with glycan structure due to differences in ionization efficiency associated with sialylation and antennary complexity. Protease digestion conditions were also optimized, and chymotrypsin was most effective for site-specific glycopeptide recovery. Distinct glycan distributions were observed between the two glycosylation sites, underscoring the functional importance of site-specific profiling. Furthermore, molecular modeling revealed that the additional glycan at Asn25 may enhance structural stability and receptor-binding affinity by reducing aggregation and strengthening interactions with interferon alpha/beta receptor subunit 2. Together, this study presents a refined approach for glycan analysis in multi-site glycoproteins, facilitating accurate assessment of glycosylation-related critical quality attributes in biotherapeutic development.<b>Keywords: </b>Interferon beta; N-linked glycan; Site-specific glycosylation; Relative quantitative analysis; R27T