The serine/threonine kinase glycogen synthase kinase-3 beta (GSK3b) is a known master regulator for several cellular pathways that include insulin signaling and glycogen synthesis, neurotrophic factor signaling, Wnt signaling, neurotransmitter signaling and microtubule dynamics. Consequently, this enzyme has been implicated in multiple human disorders including Alzheimer’s disease, bipolar disorder, noninsulin-dependent diabetes mellitus, cardiac hypertrophy, and cancer. Precisely how GSK3b maintains its pathway specificity efficiently at the crossroads of many cellular processes is still unclear. Many inhibitors of GSK3b exist, however these compounds have been found lacking in selectivity, with CHIR 99021 considered most potent and selective. The goal for this project is to identify potent and highly selective small molecule probes to investigate GSK3b biology in cellular and ultimately in whole animal models. A library of over three hundred twenty thousand compounds was screened against human GSK3b. Among the inhibitors identified, CID 5706819 showed decent potency and excellent selectivity inhibiting only five out of over three hundred kinases at 10 μM by over fifty percent. Subsequent chemical modifications of CID 5706819, guided by a co-crystal structure with GSK3b, and a battery of biochemical and cell-based assays led to CID 56840716/ML320 that inhibits GSK3b at IC50 between 10–30 nM. ML320 has a superior kinome-wide selectivity profile compared to CHIR99021. Further, ML320 demonstrates excellent cellular activity in inhibiting GSK3b-mediated Tau phosphorylation in SH-SY5Y neuroblastoma cells (IC50 of 1 μM), and in relieving negative regulation by GSK3b on cellular beta-catenin degradation and TCF/LEF promoter activities with EC50 of 5 μM in both assays. At the same time, no cellular toxicity by ML320 was observed in SH-SY5Y cells at the highest testing concentration of 30 μM. Taken together, ML320 is a potent and highly selective small molecular probe against GSK3b, allowing better investigation and interpretation of GSK3b cellular functions than the existing prior art. ML320 scaffold also holds the promise to deliver additional compounds with further improved biochemical, cellular, and pharmacokinetic properties suitable for investigating in vivo roles of GSK3b in pertinent animal physiology and pathology.