The development and deployment of monoclonal antibodies (mAbs) for therapeutic applications hinge on meticulous functional characterization to ensure efficacy and safety. This critical review delves into the characterization processes for a GLP-1 analog used in diabetes treatment and a high molecular weight mAb for arthritis. Despite promising clinical outcomes, these biologics encounter significant challenges, such as proteolytic degradation and immunogenic responses. Binding assays, activity assessments, and in vivo studies offer insights into their therapeutic mechanisms, yet discrepancies between laboratory and clinical responses highlight the need for rigorous assay optimization. Addressing these challenges involves innovative approaches and robust validation protocols. For GLP-1 analogs, detailed binding assays are essential to confirm receptor specificity and activity, while in vivo studies are critical for understanding pharmacokinetics and pharmacodynamics. For high molecular weight mAbs targeting arthritis, overcoming immunogenicity requires advanced engineering techniques to enhance biocompatibility and reduce adverse reactions. The continuous advancement in mAb development is propelled by these innovative methodologies, ensuring that new therapeutics meet the stringent requirements for clinical success. Ultimately, this progress enriches the treatment landscape for various diseases, offering improved outcomes for patients and expanding the potential of biologic therapies. Through ongoing research and development, the characterization and optimization of mAbs remain pivotal in translating laboratory discoveries into effective clinical treatments.