Developmental neurotoxicity (DNT) entails the toxic effects imparted by many chemicals on brain during early childhood. Various chemicals would have their maximum effects on brains during early childhood. Some toxicants have confirmed to induce developmental toxic effects on CNS. Most of agents cannot be identified with certainty due to the defective nature of predictive toxicology models used. A novel method that can overcome most of the limitations of conventional techniques is the use of 3D neurospheres system (in-vitro system can recapitulate most of the changes during the period of brain development making it an ideal model for predicting neurotoxic effects). In the present study we verified the possible DNT of epoxomicin which is a potent anti-tumor agent isolated from Actinomycetes that is used as a selective and irreversible inhibitor of the 20S proteasome with anti-inflammatory activity. Methods Rat neural progenitor cells were isolated from rat embryos (E14) extracted from placental tissue. Cortices were aseptically dissected from brains of the fetuses and the tissues were triturated by repeated passage through a fire-polished constricted Pasteur pipette. The dispersed tissues were allowed to settle for 3 min. The supernatant was then transferred to a fresh tube and centrifuged at 1,000 g for 5 min. The pellet was placed in Hank’s balanced salt solution cultured as free-floating neurospheres in proliferation medium. Two doses of epoxomicin (1µM and 10µM) were used in cultured neurospheres for a period of 14 days. For proliferation analysis, spheres were cultured in proliferation medium. After 0,4,5,11 and 14 days, sphere size was determined by software analyses (CellProfiler, Broad Institute, www.cellprofiler.org) Diameter of each neurosphere was measured and exported as excel file to statistical analysis. For viability analysis, trypsin-EDTA solution were added to neurospheres for 3 min to dissociate them into single cells suspension, then viability was evaluated by the Trypan Blue exclusion test. Epoxomicin found to affect differentiation and viability of neurospheres, these effects were positively correlated to doses and progress of time. Results Epoxomicin found to affect proliferation and viability of neuropsheres. Conclusion The study confirms DNT of epoxomicin and suggests possible gross morphologic changes and the decrease in viability, it proposes possible focal lesions on exposure to epoxomicin in early childhood.