© 2014 Society for Laboratory Automation and Screening. Alzheimer disease (AD) is the most common neurodegenerative disease, and with Americans' increasing longevity, it is becoming an epidemic. There are currently no effective treatments for this disorder. Abnormalities of Tau track more closely with cognitive decline than the most studied therapeutic target in AD, amyloid, but the optimal strategy for targeting Tau has not yet been identified. On the basis of considerable preclinical data from AD models, we hypothesize that interactions between Tau and the Src-family tyrosine kinase, Fyn, are pathogenic in AD. Genetically reducing either Tau or Fyn is protective in AD mouse models, and a dominant negative fragment of Tau that alters Fyn localization is also protective. Here, we describe a new AlphaScreen assay and a live-cell bioluminescence resonance energy transfer (BRET) assay using a novel BRET pair for quantifying the Tau-Fyn interaction. We used these assays to map the binding site on Tau for Fyn to the fifth and sixth PXXP motifs to show that AD-associated phosphorylation at microtubule affinity regulating kinase sites increases the affinity of the Tau-Fyn interaction and to identify Tau-Fyn interaction inhibitors by high-throughput screening. This screen has identified a variety of chemically tractable hits, suggesting that the Tau-Fyn interaction may represent a good drug target for AD.