<p>Cell migration assays provide invaluable insights into fundamental biological processes. In a companion Review, we describe commercial and custom in vitro and in vivo assays to measure cell migration and provide guidelines on how to select the most appropriate assay for a given biological question. Here, we describe the fundamental principles of how to compute—from the raw data generated by these assays—quantitative cell migration parameters that help determine the biophysical nature of the cell migration, such as cell speed, mean-squared displacement, diffusivity, persistence, speed and anisotropy, and how to quantify cell heterogeneity, with practical guidance. We also describe new imaging and computational technologies, including AI-based methods, which have helped establish fast, robust and accurate tracking of cells and quantification of cell migration. Taken together, these Reviews offer practical guidance for cell migration assays from conception to analysis.</p>