iPSC-derived human neurons are expected to revolutionize studies on brain diseases, but their functional heterogeneity still poses a problem. Key sources of heterogeneity are the different cell culture systems used. We show that an optimized autaptic culture system, with single neurons on astrocyte feeder islands, is well suited to culture, and we analyze human iPSC-derived neurons in a standardized, systematic, and reproducible manner. Using classically differentiated and transcription factor-induced human glutamatergic and GABAergic neurons, we demonstrate that key features of neuronal morphology and function, including dendrite structure, synapse number, membrane properties, synaptic transmission, and short-term plasticity, can be assessed with substantial throughput and reproducibility. We propose our optimized autaptic culture system as a tool to study functional features of human neurons, particularly in the context of disease phenotypes and experimental therapy.