Three-dimensional (3D) cell culture models are attracting significant attention by the researchers, especially in cancer therapy, as two-dimensional models have failed to replicate the 3D complexity of an in vivo tumor. Different 3D cell culture models available, including spheroids, polymer/ hydrogel scaffolds, hanging drop approach, bioreactors, organs-on-a-chip, and 3D bioprinting structures, are highlighted in this chapter. In drug discovery, new compounds are routinely screened for their anticancer efficacy using traditional 2D cell-based assays. A wide range of 3D cell culture models have been developed due to recent advances in cell biology, microfabrication methods, and tissue engineering. Scaffolds provide physical support for 3D cell culture growth. With recent advances in microfluidic fabrication technology, extensive and controlled 3D cell culture models have been designed to allow accurate microenvironmental parametric control by using biocompatible microfluidic chips fabricated by technologies, including photolithography and soft lithography.