This study outlines advanced methods for characterizing and differentiating sulphated glycan molecules isolated from seaweed extracts. A significant challenge in the field remains accurately identifying and validating functional molecular traits such as molecular weight, degree of sulfation, monosaccharide composition, protein presence, and glycosidic linkages. This work specifically addresses the development of robust and precise analytical techniques to reliably differentiate polysaccharide-rich and protein-rich fractions based on these molecular and biochemical properties. Characterization techniques employed include carbohydrate quantification using Water-Soluble Tetrazolium (WST-1) dye assays, protein estimation via Bicinchoninic Acid (BCA) assays, and polysaccharide sulfation assessments with 1,9-Dimethyl-Methylene Blue (DMMB) assays. Extract cytotoxicity was evaluated using resazurin-based cell viability assays. Gel-based analyses, such as SDS-PAGE, were utilized for protein profiling, while comprehensive compositional analyses, including gas chromatography–mass spectrometry (GC-MS) for monosaccharide and glycosidic linkage characterization, amino acid analysis, and proteomic profiling, further substantiated the validity and effectiveness of the characterization approach. Results highlighted clear differences in molecular weight and sulfation between polysaccharide-rich and protein-rich fractions, demonstrating the robustness of these methodologies in addressing complex characterization challenges.