Purpose <p>Insulin glargine is a widely used long-acting insulin analog for the management of diabetes mellitus; however, the development of anti-drug antibodies (ADAs) against glargine can compromise therapeutic efficacy and alter pharmacodynamic responses. Therefore, sensitive and reliable detection of anti-glargine antibodies is essential for effective immunogenicity monitoring. This study aimed to develop and validate a robust, sensitive, and cost-effective bridging ELISA method for the detection of anti-glargine antibodies in human serum.</p> Methods <p>A bridging ELISA was developed using insulin glargine as both capture and detection reagent. Microplates were coated with glargine (2.5&#xa0;µg/mL), followed by incubation with serum samples mixed in a 1:1 ratio with biotin-labeled glargine. Detection was carried out using streptavidin–HRP conjugate (1:40,000 dilution) and TMB substrate, with absorbance measured at 450&#xa0;nm. The assay was validated for sensitivity, specificity, signal-to-noise ratio, and dynamic range in accordance with immunogenicity assay validation principles.</p> Results <p>The validated assay demonstrated high sensitivity and specificity, with reliable detection of anti-glargine antibodies across a relative concentration range corresponding to 78 ng/mL to 5000 ng/mL (quasi-quantitative scale) at a minimum required dilution (MRD) of 1:3. The method exhibited a strong signal-to-noise ratio, enabling clear discrimination between positive and negative samples. The assay performance confirmed its suitability for accurate and reproducible detection of anti-glargine antibodies in human serum.</p> Conclusion <p>The developed and validated bridge ELISA provides a sensitive, specific, and reproducible method for monitoring anti-glargine antibodies. This assay offers a practical tool for immunogenicity assessment in clinical and research settings, supporting improved therapeutic monitoring and management of insulin glargine treatment.</p>