New discoveries in RNA biology underscore a need for chemical tools to clarify their roles in pathophysiological mechanisms. In certain cancers, synthesis of the let-7 microRNA tumor suppressor is blocked by an RNA binding protein (RBP) Lin28, which docks onto a conserved sequence in let-7 precursor RNA molecules and prevents their maturation. Thus, the Lin28/let-7 interaction might be an attractive drug target, if not for the well-known difficulty in targeting RNA-protein interactions with drugs. Here, we describe a protein/RNA FRET assay using a GFP-Lin28 donor and a black-hole quencher (BHQ)-labeled let-7 acceptor, a fluorescent protein/quencher combination which is rarely used in screening despite favorable spectral properties. We tested 16 000 molecules and identified N-methyl-N-[3-(3-methyl[1,2,4]triazolo[4,3-b]pyridazin-6-yl)phenyl]acetamide, which blocked the Lin28/let-7 interaction, rescued let-7 processing and function in Lin28-expressing cancer cells, induced differentiation of mouse embryonic stem cells, and reduced tumor-sphere formation by 22Rv1 and Huh7 cells. A biotinylated derivative captured Lin28 from cell lysates consistent with an on-target mechanism in cells, though the compound also showed some activity against bromodomains in selectivity assays. The Lin28/let-7 axis is presently of high interest not only for its role as a bistable switch in stem-cell biology but also because of its prominent roles in numerous diseases. We anticipate that much can be learned from the use of this first reported small molecule antagonist of Lin28, including the potential of the Lin28/let-7 interaction as a new drug target for selected cancers. Furthermore, this approach to assay development may be used to identify antagonists of other RBP/RNA interactions suspected to be operative in pathophysiological mechanisms.