Abstract: The confirmation of target engagement for candidate drugs is a fundamental requisite in drug discovery. A significant portion of projects fail to reach the clinic due to lack of efficacy or failure to show lead candidates interacting with the intended target in a more complex environment. The utility of using CETSA® technology to measure target engagement in intact cells is increasingly exploited in high throughput screening, examples demonstrate low false positive rates (Rowlands et al. 2023 PMID: 36736830). Here, using our newly developed high-throughput screening platform, the analysis of a high throughout plate based CETSA® HT screen completed using Alpha SureFire® technology on the well-known transcription factor target p53 is demonstrated. Hits identified from this screen are privileged from the onset by binding to the target in a physiologically relevant cellular matrix and in a binding-mode agnostic way. Unlike traditional screening methods targeting activity or invitro interaction where cellular activity is addressed in later stages. This assay cascade highlights a rapid target-to-hit lead generation strategy placing the emphasis on finding chemical matter that interacts with endogenous target in living cells first, followed by analysis of hit material activity. The ‘shortcut’ to chemical matter active in living cells represents significant potential cost savings in comparison to assay development time on recombinantly produced proteins and/or modified cell lines for traditional HTS screening approaches that still rely on target engagement assessment downstream.