MOTIVATION: During development, progenitor cells undergo multiple rounds of cellular divisions during which transcriptional programs must be faithfully propagated. Investigating the timing of transcriptional activation, which is a highly stochastic phenomenon, requires the analysis of large amounts of data. In order to perform automatic image analysis of transcriptional activation, we developed a software that segments and tracks both small and large objects, leading the user from raw data up to the results in their final form. RESULTS: MitoTrack is a user-friendly open-access integrated software that performs the specific dual task of reporting the precise timing of transcriptional activation while keeping lineage tree history for each nucleus of a living developing embryo. The software works automatically but provides the possibility to easily supervise, correct and validate each step. AVAILABILITY AND IMPLEMENTATION: MitoTrack is an open source Python software, embedded within a graphical user interface (download here). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.