CellProfiler 2.0 is an improved version of the open-source, modular high-throughput image analysis software designed to meet the growing needs of the biology research community. The new version offers enhanced robustness, user-friendliness, and functionality, including new algorithms and features for high-throughput work. Key improvements include a redesigned infrastructure using Python and high-performance scientific libraries, improved user interface with drag-and-drop operations, context-sensitive menus, and extensive help, and new and improved algorithms for tasks such as neuron image analysis, time-lapse object tracking, and illumination correction. CellProfiler 2.0 also supports batch processing, metadata loading, and integration with other open-source software, making it more versatile and efficient for large-scale image analysis projects. The software is freely available under the GPL v.2 license and can be used on various operating systems.CellProfiler 2.0 is an improved version of the open-source, modular high-throughput image analysis software designed to meet the growing needs of the biology research community. The new version offers enhanced robustness, user-friendliness, and functionality, including new algorithms and features for high-throughput work. Key improvements include a redesigned infrastructure using Python and high-performance scientific libraries, improved user interface with drag-and-drop operations, context-sensitive menus, and extensive help, and new and improved algorithms for tasks such as neuron image analysis, time-lapse object tracking, and illumination correction. CellProfiler 2.0 also supports batch processing, metadata loading, and integration with other open-source software, making it more versatile and efficient for large-scale image analysis projects. The software is freely available under the GPL v.2 license and can be used on various operating systems.