Kinase inhibitors capable of blocking the phosphorylation of protein substrates with high selectivity are essential to probe and elucidate the etiological role of such molecules and their signalling pathways. By addressing these biochemical questions in disease relevant cell-based and in vivo models, strong pharmacological evidence can be generated towards validating or disproving a target hypothesis. Pharmacological studies can also provide fundamental information to identify appropriate biomarkers and rational drug combination strategies and thereby facilitate clinical translation. However, due to the high number of kinases encoded by the human genome (>500) and their highly conserved catalytic domains, the development of such an elite class of inhibitors—a.k.a. high-quality chemical probes—represents a major challenge. Through a ligand-based inhibitor design, focused library synthesis and phenotypic screening to prioritize compounds with potent cell activity, we recently identified a cell cycle inhibitor with micromolar potency that inhibits mTOR kinase activity. Following a rapid lead optimization campaign, we report the development of eCF309, an mTOR inhibitor displaying low nanomolar potency both in vitro and in cells and an excellent selectivity profile (S-score (35%) = 0.01 at 10 μM).