Spot-On is a free, easy-to-use, drag-n-drop web application. Spot-On allows you to analyze single particle tracking datasets. Spot-On fits a realistic kinetic model to the jump length distribution of the observed trajectories and provides estimates of the bound fraction and diffusion coefficients for either a two state (bound-free) or a three state (bound-free1-free2) model. Full documentation can be found here. Spot-On was initially written by Anders Sejr Hansen from the Tjian-Darzacq Lab at UC Berkeley as a Matlab script. Maxime Woringer then ported it to Python and designed the web interface, with help and feedback from the Imaging and Modeling Lab (Institut Pasteur, Paris) and from the Tjian-Darzacq Lab (UC Berkeley).