Background: Three-dimensional (3D) in vitro cultures can recapitulate the physiological in vivo microenvironment. 3D Modeling techniques have been investigated and applied in anticancer drug screening. Materials and Methods: A silicate fiber scaffold was used for 3D cell cultures, and used to model the efficacy of anticancer drugs, such as mytomicin C and doxorubicin. Results: A unique 3D structure was observed in 13 human tumor cell lines on scaffold, and these cells exhibited higher drug resistance than cells in two-dimensional (2D) cultures. Furthermore, the production of lactate and expression of the nuclear factor-kappa B (NF-κB)-regulated genes B cell lymphoma-2 (BCL2), cyclooxygenase-2 (COX2), and vascular endothelial growth factor (VEGF) were higher in 3D cultures than in 2D cultures. Conclusion: These findings suggest that a 3D model using a silicate fiber scaffold can mimic features of cancer, and is also a suitable model for the evaluation of anticancer drugs in vitro.