The article discusses the transition from two-dimensional (2D) to three-dimensional (3D) cell culture techniques, highlighting their advantages and applications in various fields such as drug discovery, cancer research, and stem cell studies. 3D cell culture allows for a more accurate representation of in vivo conditions, providing insights into cell-to-cell interactions, tumor characteristics, drug responses, and metabolic profiling. Various 3D culture techniques, including scaffold-based methods (such as hydrogels, polymeric hard materials, hydrophilic glass fibers, and organoids) and scaffold-free methods (such as hanging drop microplates, magnetic levitation, and spheroid microplates), are described. These techniques offer unique advantages, such as better mimicry of the extracellular matrix (ECM) and improved cell-to-matrix interactions. The article also explores the use of 3D cell culture in drug discovery, where it enhances the efficacy and success rate of drug testing compared to traditional 2D models. Additionally, 3D culture is highlighted for its potential in stem cell research, where it improves the functionality and therapeutic potential of stem cells. The article further discusses the development of organ-on-a-chip models, which provide high-resolution, real-time imaging and allow for the study of complex physiological and pathological conditions. Finally, the article examines the role of 3D cell culture in tumor modeling and immunotherapy, emphasizing its ability to better replicate the in vivo tumor microenvironment and immune responses.The article discusses the transition from two-dimensional (2D) to three-dimensional (3D) cell culture techniques, highlighting their advantages and applications in various fields such as drug discovery, cancer research, and stem cell studies. 3D cell culture allows for a more accurate representation of in vivo conditions, providing insights into cell-to-cell interactions, tumor characteristics, drug responses, and metabolic profiling. Various 3D culture techniques, including scaffold-based methods (such as hydrogels, polymeric hard materials, hydrophilic glass fibers, and organoids) and scaffold-free methods (such as hanging drop microplates, magnetic levitation, and spheroid microplates), are described. These techniques offer unique advantages, such as better mimicry of the extracellular matrix (ECM) and improved cell-to-matrix interactions. The article also explores the use of 3D cell culture in drug discovery, where it enhances the efficacy and success rate of drug testing compared to traditional 2D models. Additionally, 3D culture is highlighted for its potential in stem cell research, where it improves the functionality and therapeutic potential of stem cells. The article further discusses the development of organ-on-a-chip models, which provide high-resolution, real-time imaging and allow for the study of complex physiological and pathological conditions. Finally, the article examines the role of 3D cell culture in tumor modeling and immunotherapy, emphasizing its ability to better replicate the in vivo tumor microenvironment and immune responses.