A series of mathematical equations are employed in the SEEKR2 milestoning methodology for calculating the mean first passage time (MFPT) and free energy related to binding for each milestone (∆G i ). This tutorial recognizes the value of revisiting essential equations from our earlier works. Previous SEEKR2 publications employ the following mathematical representations . For the sake of comprehensiveness and to ensure this tutorial remains a self-contained resource, we have chosen to present these equations again.