Tau oligomers have been shown to transmit tau pathology from diseased neurons to healthy neurons through seeding, tau misfolding, and aggregation that is thought to play an influential role in the progression of Alzheimer's disease (AD) and related tauopathies. To develop a small molecule therapeutic for AD and related tauopathies, we have developed in vitro and cellular assays to select molecules inhibiting the first step in tau aggregation, the self-association of tau into oligomers. In vivo validation studies of an optimized lead compound were independently performed in the htau mouse model of tauopathy that expresses the human isoforms of tau without inherited tauopathy mutations that are irrelevant to AD. Treated mice did not show any adverse events related to the compound. The lead compound significantly reduced the level of self-associated tau and total and phosphorylated insoluble tau aggregates. The dose response was linear with respect to levels of compound in the brain. A confirmatory study was performed with male htau mice that gave consistent results. The results validated our screening approach by showing that targeting tau self-association can inhibit the entire tau aggregation pathway by using the selected and optimized lead compound whose activity translated from in vitro and cellular assays to an in vivo model of tau aggregation.