Human induced pluripotent stem cell (iPSC)-derived neurons are being increasingly used for high content imaging and screening. However, differentiation and maturation iPSC-derived neurons is time-intensive, often requiring >8 weeks. The differentiating/maturing iPSC-derived neuronal cultures also tend to migrate and coalesce into ganglion-like clusters making single-cell analysis challenging, especially in miniaturized formats. Using our optimized extracellular matrix and low oxygen culturing conditions, we further modified neuronal progenitor cell seeding densities and feeder layer-free culturing conditions in miniaturized formats (i.e., 96 well) to decrease neuronal clustering, enhance single-cell identification and reduce edge effects usually observed after extended cell culture. Subsequent algorithm development refined capabilities to distinguish and identify single mature neurons, as identified by Calbindin and NeuN co-expression, from large cellular aggregates. Incorporation of astrocyte conditioned medium during differentiation and maturation periods significantly increased the percentage (i.e., ~10% to ~30%) of mature neurons (i.e., Calbindin+/NeuN+) detected at 4-weeks post-differentiation. This miniaturized, feeder layer-free format and image analysis algorithm provides an imaging and screening platform, which enables quantitative single-cell documentation of mature human neuron populations.