With the prospect of better translation compared to mouse systems, human IPSC-derived neuronal networks are a promising model system to overcome the valley of death in cognitive drug discovery for neurodegenerative disease. Synapse strengthening may prevent or reverse neurodegeneration. However, screening for compounds that achieve this requires a network capable of synapse strengthening and weakening that is supported by human astrocytes and microglia, i.e. a fully human model system of a minimal brain circuit.So far, convincing post-synaptic immunostains of hIPSC-derived neurons are rare, as are functional assays investigating pre- and post-synaptic function. Additionally, hIPSC-derived cultures are most often supported by rodent astrocytes or no astrocytes at all.HIPSCs can develop into iAstrocytes through forced expression of SOX9 and NFIB. We created and validated a cell line with doxycycline inducible SOX9-NFIB and found that these astrocytes support NGN2-induced iNeurons, are positive for S100B and GFAP and, in mono-cultures, show typical calcium waves when transduced with GFAP-driven GCaMP6f.In our iNeurons, we validated FM1-43 dye imaging, and automated synapse detection and analysis. FM1-43 dye, a fluorescent stryryl dye, inserts into the membrane and is taken up through synaptic vesicle recycling. Dye release kinetics following depolarization of iNeurons follow a (double) exponential decay indicating mature synaptic vesicle recycling. At a late timepoint of culture iNeurons show NMDA receptor expression co-localizing with pre-synaptic markers, indicating functional mature post-synaptic sites.This pipeline can be used to determine synapse maturity, and in the future to evaluate cognition enhancing drugs in a fully human culture system.