In sum, we have described the first total synthesis of the longest isoform of Tau using innovative protein chemistry methodologies. Our approach surpasses previous limitations of specificity and control in producing Tau, and offers a reliable, scalable route to obtain pure, full-length Tau in multimilligram quantities. By facilitating the production of Tau proteins with specific modifications, our method provides the necessary tools for systematic studies that can shed light on the role of different Tau isoforms in the pathology of neurodegenerative diseases. The successful development of this synthetic strategy also sets the stage for the future investigations of Tau post-translational modifications. This enhanced understanding could, in turn, drive the development of novel therapeutic strategies for AD. Finally, the potential for our approach extends beyond tauopathies, and the approaches developed here could be adapted to study other proteins relevant to neurodegeneration.