GoFigure2 is a cross-platform application for visualization, processing, and analysis of out-of-core multidimensional microscopy data (5D data sets). Users can visualize images, segment cells in 3d, track cells through time, and detect cell divisions to generate lineages. Results are stored in a MySQL database back-end. Once data has been processed, cell-based object features are quantified and can be used for sorting, color-coding, analysis, or exported to external tools. GoFigure2 was developed for biology research including studying the development of embryos (zebrafish and mouse), synthetic biology (signaling), and for drug screening.