Abstract <p>Target engagement (TE) assessments often come with bioanalytical challenges, particularly with low baseline level targets like cytokines and chemokines. Commercial immunoassay kits often cannot meet the requirements of sensitivity and drug interference. We present a novel methodology that significantly enhances the sensitivity of a conventional sandwich enzyme-linked immunosorbent assay (ELISA) for TE assessment. To increase sensitivity, various strategies were applied, including the combined use of U-bottomed assay plates and Intelligent Multifunctional Analytical Plates (IMAPlate™). Lowering volumes of enzyme substrate and stop solution in U-bottomed plate led to a concentrated color solution. The unique bottom-less "well" of the IMAPlate™ was utilized to measure the intensified color solution, resulting in a significantly magnified signal. The use of Pluronic^® F-127 detergent into the assay buffer system helped reduce assay background while improving sensitivity. The biotin labeling process of the detection antibody was optimized and combined with using poly horseradish-peroxidase (HRP)-streptavidin to further enhance sensitivity. To evaluate this approach, we developed a sandwich ELISA to quantify total (free and drug bound) serum human interleukin-13 (IL-13). A pair of non-competing for target binding antibodies was used. Due to IL-13's small size, steric hindrance was observed in the presence of NVS-0031 (anti-IL-13 mAb) and a saturation method to mitigate this interference was implemented. Despite an initial 40% sensitivity loss, we eventually improved sensitivity approximately 200-fold to achieve a limit of quantitation of 0.05&#xa0;pg/mL. This methodology has been effectively employed in other ELISA-based bioanalytical assays that require heightened sensitivity (data not shown), demonstrating its wide-ranging applicability.</p> Graphical Abstract <p></p>