Building a precise alternative neurotoxicological test is of great importance to respond to societal and ethical requirements. In this study, a new developmental neurotoxicity test (DNT) was established with the human neural progenitor cell line. ReNcell CX cells were exposed to neurotoxic chemicals (aphidicolin, hydroxyurea, cytosine arabinoside, 5-fluorouracil, and ochratoxin A) or non-neurotoxic chemicals (sodium gluconate, sodium bicarbonate, penicillin G, and saccharin). Propidium iodide (PI) was used to evaluate cell viability. BrdU and Ki-76 were employed to determine cell proliferation. Based on the cell viability and proliferation, mathematical models were built by linear discriminant analysis. Furthermore, the neurotoxic-considered chemicals inhibited cell cycle progression at the protein level, supporting the biomolecular rationale for the predictive model. Overall, these results show that the new test method can be used to determine the potential developmental neurotoxicants or new drug candidates.