Tau propagation is a key driver of Alzheimer’s disease (AD), yet selective markers of seeding-competent tau remain elusive. Ubiquitin phosphorylated at Serine-65 (pS65-Ub) is a PINK1-derived stress signal that accumulates in AD with elevated tau pathology, but its functional relationship to tau seeding is unknown. Here, using complementary imaging and biochemical approaches, we show that tau is covalently modified by pS65-Ub in AD brain. pS65-Ub modified tau is elevated in AD frontal cortex, enriched in Triton X-100-soluble fractions, and tightly associated with high-molecular-weight, oligomeric, and hyperphosphorylated tau. Importantly, immunodepletion of pS65-Ub from AD brain lysates nearly abolishes tau seeding in biosensor cells, indicating that tau seeding activity is associated with pS65-Ub modified species. Collectively, these findings establish pS65-Ub as a critical and highly specific marker for identifying, isolating, and eliminating pathogenic tau seeds. This strongly supports the development of pS65-Ub–targeted diagnostics and precision therapeutic approaches aimed at the selective clearance of disease-driving tau species, potentially transforming both early detection and intervention strategies for AD. To confirm tau as a pS65-Ub substrate and further characterize the species attached to pS65-Ub, we performed proteomic analyses. We carried out pS65-Ub IP after denaturation of frontal cortex lysates from five AD patients, with matched biotin labeled isotype IgG IPs as negative controls, and subjected the IP eluates to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.