...To better understand commonalities and differences between tauopathies, we report a multiplex assay combining immunoprecipitation and high-resolution mass spectrometry capable of detecting and quantifying peptides from different tau protein isoforms as well as non-phosphorylated and phosphorylated peptides, including those carrying multiple phosphorylations. We investigated the tau proteoforms in soluble and insoluble fractions of brain tissue from subjects with autopsy-confirmed tauopathies, including sporadic AD (n = 10), PSP (n = 11), PiD (n = 10), and CBD (n = 10), and controls (n = 10). Our results demonstrate that non-phosphorylated tau profiles differ across tauopathies, generally showing high abundance of microtubule-binding region (MTBR)-containing peptides in insoluble protein fractions compared with controls; the AD group showed 12-72 times higher levels of MTBR-containing aggregates. Quantification of tau isoforms showed the 3R being more abundant in PiD and the 4R isoform being more abundant in CBD and PSP in the insoluble fraction. Twenty-three different phosphorylated peptides were quantified. Most phosphorylated peptides were measurable in all investigated tauopathies. All phosphorylated peptides were significantly increased in AD insoluble fraction. However, doubly and triply phosphorylated peptides were significantly increased in AD even in the soluble fraction. Results were replicated using a validation cohort comprising AD (n = 10), CBD (n = 10), and controls (n = 10). Our study demonstrates that abnormal levels of phosphorylation and aggregation do indeed occur in non-AD tauopathies, however, both appear pronouncedly increased in AD, becoming a distinctive characteristic of AD pathology.