<p>The development of multi-specific biotherapeutics has revolutionized targeted therapy by simultaneously engaging multiple receptors or pathways, thereby enhancing therapeutic efficacy and specificity. However, evaluating the immunogenic potential of these complex molecules remains a significant challenge, particularly in the reliable detection of neutralizing antibodies (NAbs). To support the development of a bispecific biotherapeutic, we initially established a cell-based bioassay utilizing a cytotoxicity assay platform for NAb assessment. This traditional approach faced significant limitations due to severely limited drug tolerance which prevented accurate NAb classification. To address this hurdle, we developed a non-cell based competitive ligand binding (CLB) assay. The primary obstacle was the multi-transmembrane receptor target, which lacks a soluble form suitable for conventional immunoassays. We successfully addressed this challenge by leveraging a novel synthetic version of the multi-transmembrane receptor as the surrogate target. This enabled the development of a duplex competitive ligand binding assay utilizing a homogeneous bead-based AlphaLISA™ assay platform. This innovative duplex NAb assay significantly increased drug tolerance by at least 170-fold when compared to the cell-based assay, enabling sensitive and specific detection of NAb against each drug target binding domain. The AlphaLISA™ based CLB NAb exhibited negligible matrix interference and generated excellent intra-assay and inter-assay precision, with data concordant across different reagent lots and plate readers. Our results demonstrate that the AlphaLISA™ assay platform offers a robust, sensitive, and drug-tolerant alternative to traditional cell-based NAb assays. This approach provides a superior solution for assessment of NAb against multi-specific biotherapeutics targeting multi-transmembrane receptors.</p> Graphical Abstract <p></p>