The article discusses the importance of three-dimensional (3D) cell culture models in drug discovery and drug repositioning. Traditional two-dimensional (2D) cell cultures, which are widely used in high-throughput screening (HTS), do not accurately reflect the complex microenvironment of tissues, leading to a high failure rate in drug development. 3D cell cultures, on the other hand, more closely mimic in vivo conditions, making them a promising approach to improve the success rate of drug discovery. The article reviews common 3D culture techniques, including spheroids, hydrogel scaffolds, and microfluidic devices, and their applications in drug resistance and repositioning. It highlights the significance of extracellular matrix (ECM) composition, stiffness, concentration gradients, and stromal cells in influencing cell phenotype and drug response. The challenges and limitations of 3D cell culture technologies in HTS are also addressed, emphasizing the need for systems that are biologically relevant, scalable, and compatible with automated screening setups. Despite these challenges, the field of 3D cultures shows significant promise for advancing drug development and toxicity testing in various diseases.The article discusses the importance of three-dimensional (3D) cell culture models in drug discovery and drug repositioning. Traditional two-dimensional (2D) cell cultures, which are widely used in high-throughput screening (HTS), do not accurately reflect the complex microenvironment of tissues, leading to a high failure rate in drug development. 3D cell cultures, on the other hand, more closely mimic in vivo conditions, making them a promising approach to improve the success rate of drug discovery. The article reviews common 3D culture techniques, including spheroids, hydrogel scaffolds, and microfluidic devices, and their applications in drug resistance and repositioning. It highlights the significance of extracellular matrix (ECM) composition, stiffness, concentration gradients, and stromal cells in influencing cell phenotype and drug response. The challenges and limitations of 3D cell culture technologies in HTS are also addressed, emphasizing the need for systems that are biologically relevant, scalable, and compatible with automated screening setups. Despite these challenges, the field of 3D cultures shows significant promise for advancing drug development and toxicity testing in various diseases.