CellProfiler 4 is an updated version of the open-source image analysis software, designed to enhance speed, utility, and usability. The new version introduces several user interface refinements, including a more feature-rich 3D viewer, expanded figure contrast dialogs, and improved pipeline development tools. New modules have been added to expand the software's capabilities, such as the CombineObjects module for merging object sets and the RunImageJMacro module for integrating ImageJ functions. Performance improvements have been made to reduce computational time, particularly for complex workflows like 3D segmentation and the Cell Painting assay. These enhancements will make high-throughput image analysis more accessible and efficient for researchers, reducing both computational costs and environmental impact. The migration to Python 3 ensures long-term compatibility and maintainability, while the split into cellprofiler and cellprofiler-core packages lays the groundwork for a stable API for integration with other Python-based applications.CellProfiler 4 is an updated version of the open-source image analysis software, designed to enhance speed, utility, and usability. The new version introduces several user interface refinements, including a more feature-rich 3D viewer, expanded figure contrast dialogs, and improved pipeline development tools. New modules have been added to expand the software's capabilities, such as the CombineObjects module for merging object sets and the RunImageJMacro module for integrating ImageJ functions. Performance improvements have been made to reduce computational time, particularly for complex workflows like 3D segmentation and the Cell Painting assay. These enhancements will make high-throughput image analysis more accessible and efficient for researchers, reducing both computational costs and environmental impact. The migration to Python 3 ensures long-term compatibility and maintainability, while the split into cellprofiler and cellprofiler-core packages lays the groundwork for a stable API for integration with other Python-based applications.