To elucidate the role of Tau isoforms and post-translational modification (PTM) stoichiometry in Alzheimer’s disease (AD), we generated a high-resolution quantitative proteomics map of 95 PTMs on multiple isoforms of Tau isolated from postmortem human tissue from 49 AD and 42 control subjects. Although Tau PTM maps reveal heterogeneity across subjects, a subset of PTMs display high occupancy and frequency for AD, suggesting importance in disease. Unsupervised analyses indicate that PTMs occur in an ordered manner, leading to Tau aggregation. The processive addition and minimal set of PTMs associated with seeding activity was further defined by analysis of size-fractionated Tau. To summarize, features in the Tau protein critical for disease intervention at different stages of disease are identified, including enrichment of 0N and 4R isoforms, underrepresentation of the C terminus, an increase in negative charge in the proline-rich region (PRR), and a decrease in positive charge in the microtubule binding domain (MBD). Display omitted •95 post-translational modifications (PTMs) were identified on Tau in human subjects•Frequency and PTM stoichiometry of pathological Tau identify patient heterogeneity•Modifications occur in a processive fashion and are reflective of disease progression•The study identifies critical therapeutic targets at each stage of disease A high-resolution quantitative proteomics map of post-translational modifications on multiple isoforms of Tau shows heterogeneity across Alzheimer’s disease patients, reflective of disease progression, and identifies critical targets at each stage of disease.