Statement of Purpose: Researchers have shown that 3D cultures better mimic the in vivo tumor microenvironment than 2D cultures . Therefore, 3D culture of cancer cells using 3D scaffolds has been investigated to explore the interaction between cells and stroma. Composition of the 3D substrate influences cell function, signaling, and cell morphology . Chitosan (C) is a commonly used biomaterial that has beneficial biological properties including hydrophilic surface, biodegradability, and biocompatibility . Due to its cationic nature, chitosan can form an ionic complex by mixing with anionic polymers. Chondroitin sulfate (CS) is one of glycosaminoglycans in proteoglycans . CS was found in greater amounts in advanced and metastatic prostate cancer than early stage . In this study, novel 3D porous C-CS scaffolds were prepared by mixing chitosan with CS and three scaffold compositions were prepared with different CS concentrations: 0.1 wt% (4-0.1), 0.5 wt% (4-0.5) and 1 wt% (4-1) CS. The effect of CS composition in scaffolds on prostate cancer (PCa) cells was investigated by in vitro analysis with PC-3 and 22Rv1 cell lines. Our hypothesis is that PCa cultures will show higher expression of cytokeratin 8 (KRT8), androgen receptor (AR), prostate specific antigen (PSA), and LIM domain kinase 1 (LIMK1) in scaffolds with higher CS concentration.