Chemicals that specifically alter human neurite outgrowth pose a hazard for the development of the nervous system. The identification of such compounds remains a major challenge, especially in a human test system. To address this issue, we developed an imaging-based procedure in LUHMES human neuronal precursor cells to quantify neurite growth of unfixed cultures. Live imaging allowed the simultaneous evaluation of cell viability and neurite outgrowth within one culture dish. The procedure was used to test the hypothesis that inhibitors of specific pathways can impair neurite outgrowth without affecting cell viability. Although the cells were grown at high density to allow extensive networking, overall neurite growth in this complex culture was quantified with a signal-to-noise ratio of > 50. Compounds such as U0126 slowed the extension of neuronal processes at concentrations > 4 times lower than those causing cell death. High numbers of individual viable cells without neurites were identified under such conditions, and neurite outgrowth recovered after washout of the chemical. Also an extension-promoting compound, Y-27632, was identified by this unique multiparametric imaging approach. Finally, the actions of unspecific cytotoxicants such as menadione, cadmium chloride, and sodium dodecyl sulfate were tested to evaluate the specificity of the new assay. We always found a ratio of EC50 (cell death)/EC50 (neurites) < 4 for such chemicals. The described novel test system may thus be useful both for high-throughput screens to identify neuritotoxic agents and for their closer characterization concerning mode of action, compound interactions, or the reversibility of their effects.