Three-dimensional (3D) cell culture models are increasingly being used in drug discovery and drug repositioning to better mimic in vivo environments. Traditional two-dimensional (2D) cell cultures on plastic surfaces do not accurately represent the complex microenvironment of tissues, leading to high failure rates in drug development. 3D models, such as spheroids, hydrogels, and organoids, offer more accurate representations of tissue-specific environments, potentially improving drug discovery outcomes. These models can better predict drug efficacy and resistance, and are particularly useful for studying diseases like cancer, neurological disorders, and fibrotic diseases. 3D cultures can replicate ECM composition, matrix stiffness, concentration gradients, and interactions with stromal cells, which are critical factors in drug response. However, challenges remain in scaling 3D models for high-throughput screening (HTS) and ensuring compatibility with existing screening technologies. Advances in scaffold-based, synthetic, and peptide-based hydrogels, as well as microfluidic devices and organ-on-a-chip systems, are helping to overcome these limitations. 3D models, especially those derived from patient tissues, offer more accurate and personalized drug screening platforms, improving the efficiency and success of drug discovery and repositioning. Despite challenges in scalability and integration with HTS, 3D cell culture technologies are becoming essential tools in modern drug development.Three-dimensional (3D) cell culture models are increasingly being used in drug discovery and drug repositioning to better mimic in vivo environments. Traditional two-dimensional (2D) cell cultures on plastic surfaces do not accurately represent the complex microenvironment of tissues, leading to high failure rates in drug development. 3D models, such as spheroids, hydrogels, and organoids, offer more accurate representations of tissue-specific environments, potentially improving drug discovery outcomes. These models can better predict drug efficacy and resistance, and are particularly useful for studying diseases like cancer, neurological disorders, and fibrotic diseases. 3D cultures can replicate ECM composition, matrix stiffness, concentration gradients, and interactions with stromal cells, which are critical factors in drug response. However, challenges remain in scaling 3D models for high-throughput screening (HTS) and ensuring compatibility with existing screening technologies. Advances in scaffold-based, synthetic, and peptide-based hydrogels, as well as microfluidic devices and organ-on-a-chip systems, are helping to overcome these limitations. 3D models, especially those derived from patient tissues, offer more accurate and personalized drug screening platforms, improving the efficiency and success of drug discovery and repositioning. Despite challenges in scalability and integration with HTS, 3D cell culture technologies are becoming essential tools in modern drug development.