Understanding a molecule's mode of action is crucial in the development of chemical probes and the broader drug discovery process. The Cellular Thermal Shift Assay (CETSA) stands out as an essential technique for verifying the engagement of a target by a small molecule within intact cells. CETSA directly measures the biophysical interactions between ligands and their protein targets, which can influence the protein's stability and aggregation under heat stress. Conventionally, CETSA experiments require separate samples for each temperature point or ligand concentration, limiting the assay's throughput and demanding significant optimization. To capture the full aggregation profile of a protein from a single sample, we developed a prototype real-time CETSA (RT-CETSA) platform that couples a real-time PCR instrument with a CCD camera to detect luminescence during a melting experiment. Concurrently, we engineered a thermally stable NanoLuciferase variant (ThermLuc), capable of maintaining structural integrity at temperatures exceeding 90°C, proving its efficacy in tracking target engagement across various proteins. Utilizing well-characterized inhibitors of lactate dehydrogenase alpha, RT-CETSA showed significant correlation with enzymatic, biophysical, and other cell-based assays. A data analysis pipeline was developed to enhance the sensitivity of RT-CETSA in detecting on-target binding. Collaborating closely with Arimation Robotics, we refined the prototype design and developed a built-for-purpose device towards creating a RT-CETSA technology package that could be adopted by the broader scientific community. The resultant RT-CETSA platform significantly advances the capabilities of the CETSA method, facilitating the identification of ligand-target engagement in cells, a critical step in assessing the mechanism of action of a small molecule.