Neurodegenerative diseases, collectively known as tauopathies, are characterized by intra-neuronal deposits of a microtubule-associated protein Tau (MAPT). Alzheimer's disease (AD), the leading cause of dementia and a representative tauopathy, is marked by the presence of senile plaques and neurofibrillary tangles composed of hyperphosphorylated Tau. The protein Tau, first described by Kirschner, belongs to a family of microtubule-associated proteins. Tau is expressed in six variants resulting from alternative pre-splicing of a single MAPT gene (Figure ). These isoforms differ by the presence of one/two inserts in the N-terminal fragment and repeat regions (R) in the microtubule-binding domain (MTB) of the Cterminus (Figure ). As an intrinsically disordered protein, Tau is highly susceptible to post-translational modifications (PTMs, Figure ). These modifications, including phosphorylation, acetylation, glycosylation, ubiquitination, SUMOylation, and methylation play crucial roles in regulating Tau localization, protein-protein interactions, degradation/clearance, and microtubule binding. Importantly, PTMs alter the charge and hydrophobicity, potentially providing the driving force for Tau to undergo aggregation associated with neurodegenerative diseases. Various strategies have been employed to examine the role PTMs in Tau biology. One such method is isolating Tau from post-mortem samples or from tauopathy mouse models. Another common method involves enzymatic functionalization of Tau to obtain phosphorylated, acetylated, ubiquitinated, and glycosylated forms, but this method lacks specificity and control over the location or extent of modifications. Furthermore, preparation of pure Tau is challenging because, as a disordered protein, Tau is vulnerable to protease degradation. While more site-specific methods exist, they rely on the use of point mutations that mimic charge-neutralizing PTMs (S to D mutations for phosphoserine, or K to Q for acetyl lysine ) or by using bioconjugate functionalization of dehydroalanine. The major disadvantage of this strategy is that these mimics may not possess the same structure, acidity, or charge as the natural PTMs. Chemical synthesis, on the other hand, may overcome all these limitations because it enables the preparation of the exact same chemical structure as patients with complete specificity. Semi-synthesis which combines recombinant proteins with chemical synthesis has been exploited by several groups, including studies on hyperphosphorylation/O-glycosylation in the C-terminus (Hackenberger), phosphorylation and acetylation in the microtubule binding domain (Lashuel), and lysine carboxymethylation (Becker). These studies have provided important insights into the role of individual PTMs mostly located in the MTB or C-terminal region and complement chemical approaches to understand the molecular mechanisms of neurodegeneration based on perturbation of protein structure/function in Parkinson's disease (a-synuclein with various PTMs such as nitration, phosphorylation, N-terminal acetylation, O-GlcNAc, and ubiquitination ), AD (Ab), and Huntington's disease (huntingtin). Despite abovementioned efforts, complete chemical synthesis of full-length Tau has remained elusive. We posited that total synthesis of Tau can overcome these limitations, enabling the production of the same chemical structure found in patients with complete specificity. Given the broad structural diversity and localization of PTMs throughout Tau, a chemical strategy that can systematically evaluate each PTM individually, or in combinations, would greatly facilitate in vitro investigations into the role of site-specific PTMs. Here, we report the first total synthesis of the longest isoform of Tau (2N4R) optimized to minimize the number of disconnections without compromising efficiency, chemical yield, and purity.