A three-dimensional spheroid cell culture model resembles the architecture and cellular characteristics of solid tumors much more closely than the conventional monolayer cell cultures often applied in preclinical drug testing. In this study, we validate the efficacy of the RIST treatment protocol, a novel multimodal treatment regimen for high-risk neuroblastoma, in a neuroblastoma spheroid cell culture model characterized by an augmented neoplastic phenotype. The results also indicate the importance of the neuroblastoma spheroid model for preclinical drug testing in a rigorous, robust and efficient high-throughput format, which can be beneficial to identify more effective treatment options for children with high-risk neuroblastoma in the future. Background: The outcome for patients with high-risk neuroblastoma remains poor and novel treatment strategies are urgently needed. The RIST protocol represents a novel metronomic and multimodal treatment strategy for high-risk neuroblastoma combining molecular-targeted drugs as 'pre-treatment' with a conventional chemotherapy backbone, currently evaluated in a phase II clinical trial. For preclinical drug testing, cancer cell growth as spheroid compared to mo-nolayer cultures is of advantage since it reproduces a wide range of tumor characteristics, including the three-dimensional architecture and cancer stem cell (CSC) properties. The objective of this study was to establish a neuroblastoma spheroid model for the rigorous assessment of the RIST treatment protocol. Methods: Evaluation of CSC marker expression was performed by mRNA and protein analysis and spheroid viability by luminescence-based assays. Aberrant expression of RNA-binding protein La in neuroblastoma was assessed by tissue microarray analysis and patients' data mining. Results: Spheroid cultures showed increased expression of a subgroup of CSC-like markers (CXCR4, NANOG and BMI) and higher Thr389 phosphorylation of the neuroblastoma-associated RNA-binding protein La when compared to monolayer cultures. Molecular-targeted 'pre-treatment' of spheroids decreased neoplastic signaling and CSC marker expression. Conclusions: The RIST treatment protocol efficiently reduced the viability of neuroblastoma spheroids characterized by advanced CSC properties.