Despite advances in multimodal treatment, neuroblastoma (NB) is often fatal for children with high-risk disease. There is an urgent need to develop new drugs which can be fast-tracked into frontline therapy to improve cure rates for patients with high-risk disease and to decrease toxicity in long-term survivors. MYCN amplification and anaplastic lymphoma kinase (ALK) activation have been identified as two major oncogenic events in NB pathogenesis, especially in the high-risk group. Our aim was to develop and validate pharmacodynamic (PD) biomarkers to evaluate both proof of mechanism and proof of concept for drugs that block such as PI3K/AKT/mTOR or ALK pathway activity in children with neuroblastoma. In this study, we hypothesized that screening a small-molecule library might identify already existing drugs that are able to modulate the oncogenic activity, specifically found in NB. To identify small molecules with the potential of inhibiting oncogenic activity and, consequently, its downstream genes such as MYCN or ALK, we implemented a high throughput chemical screen, using a curated library of ~450 compounds from the Scientific Research on Innovative Areas, Scientific Support Programs for Cancer Research, from The Ministry of Education, Culture, Sports, Science and Technology, Japan. In the drug screen, aurora kinase inhibitors (three molecules), JAK-STAT kinase inhibitor (one molecule) and proteasome inhibitor, in particular the proteasome-selective compound, bortezomib, was the most discriminatory with regard to sensitivity for MYCN-amplified cell lines. In an expanded panel of 20 NB cell lines, those with or without MYCN-amplification or 11q loss of heterozygousity were the most sensitive to low nanomolar concentrations of those compounds. We demonstrated that novel candidate compounds were rapidly and effectively identified by an in screening strategy, followed by in vitro assays. Therefore, our studies have uncovered a novel targetable susceptibility in high-risk neuroblastoma with potential clinical application.