Neurons derived from human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), are excellent resources for disease modeling and drug screening. Human PSCs derived neural stem cells (NSCs) can be expanded in culture and further differentiated into mature neurons for various applications, however, these often contain mixed population of both differentiated neurons and undifferentiated NSCs. Due to the continuing proliferation of undifferentiated NSCs, very high cell densities and cell aggregation are usually observed during the differentiation of hPSC-derived NSCs which increase over time, posing challenges for long-term maintenance and downstream analysis. Here we demonstrate the use of a new supplement which can reduce the proliferation of undifferentiated NSCs without negatively impacting the rate or extent of differentiation for hPSC-derived NSCs. The overall effect increases the relative population of differentiated neurons in culture. Typically under these conditions by 2-3 weeks, differentiated neurons with extensive neurite networks are seen that are evenly distributed across the culture surface, with very little clumping or aggregation observed. Further, this more uniform morphology and enriched neuron population greatly facilitates quantitative image analysis, as demonstrated by high content analysis using automated a CellInsight™ CX5 imaging platform which showed differentiated neurons expressing mature neuronal markers MAP2 or HuC&D without the contamination of undifferentiated Nestin positive NSCs, as seen in control samples. Multielectrode array (MEA) analysis demonstrates that differentiated neurons fired spontaneous action potentials, indicating functional neurons. In addition, use of this new supplement enabled differentiated neurons to be maintained for longer time in culture than untreated control cells.