There is an increased need to develop reliable and efficient screening tools to identify chemicals with the potential to adversely affect neurological development. Human iPSC-derived neurons exhibit the functionality and behavior of mature neurons and are available in quantities sufficient for screening workflows. We have developed imaging and analysis methods that provide tools for the assessment of multiple toxicity phenotypes using live neuronal cells. Phenotypic read-outs included characterization of neurite outgrowth, branching, number of processes, cell viability, and nuclear condensation. Since mitochondrial function has been associated with neuronal function we have tested changes of mitochondria membrane potential. In addition, we have evaluated processes of autophagy and mitophagy, characterized by co-localization of mitochondria and autophagy granules. Autophagosomes can be readily visualized and characterized even in heterogeneous cell populations using automated imaging and high-content analysis of live or fixed cells. Additional information can be obtained about the mechanisms of autophagic stimulation or inhibition by studying the co-localization of mitochondria or lysosomes with autophagosomes. Dose-response curves were generated from multiple data outputs on a cell-by-cell basis, allowing discrimination between simple cytotoxicity and specific effects on cellular function. To validate the assay, the library of 80 compounds was evaluated. The library contained compounds known to cause neurotoxicity, compounds of environmental concern, and compounds that serve as negative controls. Effects on neurite outgrowth and viable cell number were assessed following a 72 hr of exposure. Mitochondrial membrane potential (MMP) activity was evaluated in parallel following a 1 hour exposure. Compounds were ranked by activity and selectivity (i.e., compound specific effects on neurite outgrowth and mitochondria integrity independent of cytotoxicity). This strategy is useful for ranking compounds for potential neurotoxicity effects and aids in prioritization for more comprehensive in vivo assessments.