Three-dimensional (3D) cell culture allows cells to growth in their 3D physical shape and interact with their surroundings which represent the natural microenvironment. Hydrogels are crosslinked networks, have become increasingly used biomaterial for 3D cell culture. In this thesis, a new methodology based on bio-patterning was developed to fabricate (3D) cellular structures by using Mg-alginate hydrogel and fabricated 3D cellular structures was utilized for drug screening studies. Mg-alginate hydrogel has a specific gelation/de-gelation characteristics compared to other types of hydrogels due to its weak polymer-ion interaction. In this study, slow gelation and de-gelation property of Mg-alginate hydrogel was used for biopatterning of 3D cellular structures. Plackett-Burman and Box-Behnken design models were used to optimize parameters of Mg alginate-based biopatterning method while using HeLa cells as a model cell line. The applicability of biopatterning was successfully demonstrated by using SaOS-2 and SH-SY5Y cells. Cell proliferation and viability of long-term cultured tumor models were analyzed and immunostaining was done to investigate cellular and extracellular components of 3D cellular structures. The dose-response of fabricated 3D structures was evaluated and compared with standard 2D cell culture by applying doxorubicin (DOX). The IC50 values were calculated for 3D cellular structure of HeLa, SaOS-2 and SH-SY5Y cells as 8.2, 7.8, and 2.1 μM respectively while IC50 values of 2D controls obtained as 3.2, 4.4, and 0.2 μM respectively. These results were also statistically analyzed and dose responses were found significantly different according to t-test, which means 3D cellular structures were more resistant to drug exposure compared to 2D cell culture.