Spontaneous neuronal activity (SNA) plays an important role in the maturation and refinement of neuronal connections. However, neurodegenerative diseases, like tauopathies, can significantly alter SNA. Changes in SNA after the expression of tau mutations have not yet been analysed at presymptomatic stages. Furthermore, the currently available models lack the necessary complexity of the human brain to fully understand the underlying mechanisms of tau pathology and its effects on SNA. To address this, we present a novel in vitro platform utilizing cortical organoids (COs), derived from hPSCs, to unravel the effects of tau pathology on SNA by exploring the changes that occur after the inclusion of mutated and non-mutated tau.We have characterised COs using both commercial and healthy donor-derived hPSCs. To model tauopathy, COs were infected with adeno-associated virus that express either mutated P301L-tau or full-length non-mutated human tau (2N4R). We employed calcium imaging techniques using genetically encoded calcium indicators and two-photon microscopy recordings to analyse changes in SNA patterns and their correlation with tau pathology.Through overexpression of P301L-tau, we successfully developed COs that exhibit hyperphosphorylated tau as observed by biochemical analysis of phospho-tau(Ser422) and AT8, as well as tau aggregates observed by positive staining for thioflavin-S. Additionally, we have shown that these COs have SNA with observed changes in neuron firing frequency between P301L-tau, 2N4R-tau, and uninfected COs.Our findings suggest that tau mutations alter SNA of COs and that the model can be used to further explore the functional consequences of tau mutation-mediated changes in SNA.