Abstract Neuroblastoma is the most common pediatric malignancy with poor response to current therapy. The NF-kB/mTOR, hedgehog and PLK1 pathways/molecules are aberrantly expressed and activated in high-risk neuroblastoma, thereby targeting these pathways/molecules is an attractive therapeutic strategy. Therefore, we investigated the efficacy and associated molecular mechanism(s) of NF-kB/mTOR dual inhibitor 13-197, hedgehog inhibitor Vismodegib and PLK1 inhibitor BI2536 alone or in combination with Topotecan against high-risk neuroblastoma in vitro and in vivo. Using three neuroblastoma cell lines, the in vitro efficacy of the inhibitors as single agents or in combination with Topotecan on cell growth and apoptosis along with associated molecular mechanism(s) were investigated. In addition, the combined efficacy of Vismodegib and Topotecan was determined in vivo using a xenograft mouse model. Results showed that 13-197, BI2536 and Vismodegib significantly decreased neuroblastoma cell growth and induced apoptosis by targeting associated pathways and molecules in vitro. The 13-197 and BI2536 as single agents showed similar efficacies and were most potent in inhibiting cell growth and survival of neuroblastoma cells. In combination with Topotecan, BI2536 or Vismodegib further significantly decreased neuroblastoma cell growth/survival and neurospheres formation. Corresponding changes in the expression of targeted molecules following therapy were observed. Together, in vitro data demonstrated that hedgehog pathway inhibitor Vismodegib was most efficacious in potentiating Topotecan-induced antineuroblastoma effects. Therefore, as a next logical step, we tested the combined efficacy of Vismodegib and Topotecan against neuroblastoma in vivo using NOD-SCID Gamma deleted (NSG) mice. Our in vivo results showed that Vismodegib combined with Topotecan significantly (p<0.001) reduced tumor growth and increased survival of xenograft mice compared to single agent activity. This combination not only significantly reduced tumor growth and increased survival of the mice but also significantly enhanced antineuroblastoma efficacy by targeting hedgehog pathway components in neuroblastoma in vivo. Thus our studies lay a foundation for taking this therapeutic strategy to clinical setting to determine their efficacies in high-risk neuroblastoma patients. Citation Format: Nagendra K. Chaturvedi, McGuire R. Timothy, Don W. Coulter, Ashima Shukla, Erin M. McIntyre, J. Graham Sharp, Shantaram S. Joshi. Improved therapy for neuroblastoma using small molecule inhibitors in combination with chemotherapy. abstract. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B05.