In Alzheimer’s Disease (AD), hyperphosphorylated tau proteins are self-aggregated into paired helical filaments and accumulated into neurofibrillary tangles, which are hallmarks of AD and other tauopathies. Such amyloid filaments are toxic and tend to propagate across cells and thus are key therapeutic targets. Recent cryo-EM structures have revealed that tau adopts distinct filament conformations in the brain that are specific to disease1 and these conformations are unable to be recapitulated in vitro. Given that hyper-phosphorylation of tau is prevalent in ex-vivo amyloid filaments3, we hypothesize such differences in filament morphologies stem from the modifications, particularly phosphorylation. To test this hypothesis, I generated hyperphosphorylated tau proteins from a eukaryotic baculovirus expression system in insect cells based on the previously established method4. Then, we determined whether purified phosphorylated tau could propagate into AD-like fibrils using AD brain homogenate as a seed. Here I present the validation of phosphorylated tau proteins followed by assessment of fibril formation by kinetic assays, negative stain and immuno-electron microscopy. The results show that the phosphorylated insect cell derived tau can adopt conformation that appears like the AD fibrils.