This is an introduction for the use of our tracking software. The software can be modified and reproduced, as long as the source is cited (Careccia et al., JoVE, 2019). The software runs properly in Matlab, R2015a or later versions. Below a step by step guide of how to use it. There are three folders: "Segmentation Routines" contains the functions to be run for the segmentation. "Tracking Routines" instead contains the functions required for the tracking, most of which come from the "Simple Tracker" software from Jean-Yves Tinevez. "Example Segmentation Tracking" provides the basic scripts and files to get acquainted with the system. They allow the tracking of cells in the small multiframe tif file Example.tif. It can be easily adapted to be run in other data files. All next steps are reffered to scripts contained in "Example Segmentation Tracking" folder. First, one can use the script "DoSegmentation.m". To do so, the script has to be modified so that the name and the destination of the input .tif file are correctly stated. Similarly, the directory of the "Segmentation Routines" should be changed. The result will be saved as a file .mat, like "SegmentExample.mat". The quality of the segmentation can be checked using the script "CheckSegmentation.m", making sure that the names of the .tif and .mat files are now the proper ones. To generate the tracks, run the script "GenerateTracks.m", again modyfing it in such a way that it takes the proper segmentation file as an input, for example the "SegmentExample.mat". The quality of the tracks can be evaluated using "CheckTracking.m", again by properly changing the name of input files, directory, etc... For any querys, do not hesitate to contact me at samuel.zambrano@gmail.com. SZ, Milan, November 2018.