Cellular morphodynamics can be used as markers for many physiological and pathological processes. This protocol provides a step-by-step guide to identify variations in motility and morphology within (or across) cell populations using non-invasive live imaging and reproducible image analysis techniques such as segmentation and tracking. Detailed instructions cover all the way from cell culturing and labelling to automatic image and statistical analyses, including the definition of multiple descriptors that characterise the shape and movement of cells in a quantitative manner. All methods are available as free open-source software and illustrated by video tutorials.