Abstract Summary: We describe a modular MATLAB® Toolbox named TACTICS for time-lapse image analysis that meets several requirements not generally offered by currently available software packages: (i) the ability to assess quality of extracted imaging information by directly linking data end points to the original position, (ii) massively parallel analysis of each parameter, for flow cytometry-like assessment of possible relationships between parameters within sub-populations of the images, (iii) options for user control of the tracking such as an interface to restrict the analysis region, (iv) manual correction of automated processes and (v) user interfaces for post-tracking analysis that is linked to the original images, including options to view cell pedigrees and normalized polarization ratios based on fluorescence ratiometric measurements. Availability and implementation: We provide TACTICS source code as well as video tutorials, data sample and comprehensive user guide on the TACTICS toolbox website (http://www.tactics-toolbox.com). Installation of TACTICS requires MATLAB 7.6 (R2008a) with the presence of Image Processing Toolbox™ and Statistics Toolbox™. Contact: [email protected].