Immunological‐based assays have been widely used in diagnostic applications since the initial description of the methodology for the detection of human insulin. However, the development of immunoassays has been hindered by inherent cross‐reactivities between antibodies (Ab) and closely related antigens (Ag) that contribute to false‐positive results. In this work, we utilize temperature‐dependent immunoassay measurements to distinguish between closely related flavivirus antigens—obviating the need for a laborious development effort to ensure assay specificity. A novel platform named Thermoelectrically Activated Chips for Optical Sensing (TACOS), consisting of a miniaturized well plate overlaid on an array of thermoelectric cooling or heating pixels, enables simultaneous immunoassay measurements at different binding temperatures. The efficacy of Ab‐Ag complex formation correlates with the affinity between the Ab‐Ag pair, which is in turn dependent on the binding temperature controlled by the TACOS platform. In this study, Ab‐Ag complex formation over a temperature range is demonstrated as a unique identifier of Ab‐Ag complex composition. We contend that TACOS circumvents the need for extensive and expensive immunoassay developments and therefore enables future diagnostic tests suitable for far‐forward field applications.