Although progressive neurodegenerative diseases like Alzheimer's disease (AD) have been extensively studied for decades, some underlying mechanisms of pathogenesis remain elusive. In addition, modeling neurodegenerative diseases in vitro has proven to be a challenging task. However, advances in the technique of using human induced pluripotent stem cells (hiPSCs) have enabled the scientific community to study hiPSC-derived neurons, astrocytes, or microglia. Despite this important progress, monocultures of individual cell types may not accurately reflect the complexities of modeling specific pathological mechanisms. Therefore, we present a robust protocol for co-cultivating hiPSC-derived cortical neurons, astrocytes, and microglia in a tri-culture system for the purpose of hypothesis testing and drug screening. This co-cultivation system may allow modeling the effect of astrocyte and/or microglia modulation on neuronal health or intra-neuronal Tau aggregation, a key feature of AD progression.