Development of novel therapeutics is limited by a lack of accurate preclinical models for testing, specifically the inability of traditional 2D culture (monolayer) to accurately mimic in vivo tumors. In this work, lyophilized silk fibroin scaffolds were used to develop 3D neuroblastoma models (scaffolded NB) using multiple neuroblastoma cell lines (SK-N-AS, KELLY, and SH-SY5Y). Cells grown on scaffolds in low (1%) and ambient (21%) oxygen were compared to traditional monolayer cell culture. Monolayer cultures under low oxygen conditions exhibited increased expression of hypoxia-related genes such as VEGF, CAIX, and GLUT1. Scaffolded NB exhibited increased hypoxia-related gene expression under both low and ambient oxygen conditions. Pimonidazole staining confirmed the presence of hypoxic regions in the scaffolded NB. Cytokine secretion in the monolayer and scaffolded NB suggested differential secretion of cytokines due to both oxygen concentration (ex. VEGF, CCL3, and uPAR) and scaffolded culture (ex. IL-8, GM-CSF, and ITAC). Response to etoposide, a standard chemotherapeutic, demonstrated a reduced cytotoxicity in scaffolded culture as compared to monolayer culture regardless of oxygen concentration. However, use of a hypoxia-activated therapeutic, tirapazamine, exhibited cytotoxicity under scaffolded, ambient oxygen conditions and under monolayer and scaffolded, low oxygen conditions. Overall, this culture system provides a platform to study neuroblastoma and to assess the impact of hypoxia on tumor-relevant pathways and environments to aid in development of novel targeted therapeutics.