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Cell culture is an indispensable in vitro tool used to improve our perception and understanding of cell biology, the development of tissue engineering, tissue morphology, mechanisms of diseases and drug action. Efficient cell culturing techniques both in vitro and in vivo allow researchers to design and develop new drugs in preclinical studies. Two-dimensional (2D) cell cultures have been used since 1900s and are still a dominant method in many biological studies. However, 2D cell cultures poorly imitate the conditions in vivo. Recently three-dimensional (3D) cell cultures have received remarkable attention in studies such as drug discovery and development. Optimization of cell culture conditions is very critical in ensuring powerful experimental reproducibility, which may help to find new therapies for cancer and other diseases. In this chapter, we discuss the 2D and 3D cell culture technologies and their role in drug discovery.