Insulin-degrading enzyme (IDE) is a thiol-sensitive zinc-metallopeptidase that is strongly implicated in the pathogenesis of multiple highly prevalent diseases, including type 2 diabetes and Alzheimer’s disease (AD). Because IDE is the principal insulin-degrading protease in vivo, IDE inhibitors should enhance insulin signaling and thus have efficacy in relevant animal models of diabetes and also in therapy. Despite decades of study, a strong need yet exists for the identification of potent, selective, in vivo stable small molecule experimental probes that inhibit IDE with significantly high potency and target selectivity. We herein describe an IDE inhibitor molecular probe, ML345, which targets a specific cysteine residue (Cys819) in IDE. ML345 arose from an ultra high-throughput screening (uHTS) campaign that was supplemented with medicinal chemistry SAR optimization and with biochemical mechanistic profiling efforts. The probe is distinguished from prior art inhibitors, such as the potent and selective (but peptide-derived) hydroxamic acids and from weaker and far less selective small molecules that have been described in the literature. Probe ML345 is well-suited for use as a pharmacophore for drug development in diabetes research and as an experimental probe to understand the array of effects displayed by IDE in biological systems.