<p>Hyperphosphorylation of the microtubule-associated protein Tau is a major hallmark of Alzheimer’s disease (AD) and other tauopathies. Understanding the protein kinases that phosphorylate Tau is critical for the development of new drugs that target Tau phosphorylation. At present, the repertoire of the Tau kinases remains incomplete, and methods to uncover novel upstream protein kinases are still limited. Here, I apply our newly developed proteomic strategy, fluorescence complementation mass spectrometry (FCMS), to identify novel kinase candidates of Tau. By constructing Tau- and kinase-fluorescent fragment library, I detected 59 Tau-associated kinases, including 23 known kinases of Tau and 36 novel candidate kinases. In the validation phase using in vitro phosphorylation, 15 candidate kinases were successfully expressed and purified, and four candidate kinases, OXSR1, DAPK2, CSK, and ZAP70, displayed the ability to phosphorylate Tau. Furthermore, co-expression of these four kinases along with Tau increased the phosphorylation of Tau in human neuroglioma H4 cells. I demonstrate that FCMS is a powerful proteomic strategy to systematically identify potential kinases that can phosphorylate Tau in cells. Our discovery of new candidate kinases of Tau can present new opportunities for developing AD therapeutic strategies.</p>