Cell migration is essential to many biological processes such as embryonic development, immune surveillance and wound healing. Random cell migration refers to the intrinsic ability of cells to migrate, often called cell motility. This basal condition contrasts with directed cell migration, where cells migrate toward a chemical or physical cue. Unlike Brownian particles, however, randomly migrating cells exhibit a directional persistence, i.e., they are more likely to sustain the movement in the direction they previously took than to change, even if this direction is randomly chosen in an isotropic environment. Here we describe how to set up time-lapse recording of mammalian cells freely moving on a two-dimensional surface coated with extracellular matrix proteins, how to acquire single cell trajectories from movies and how to extract key parameters that characterize cell motility, such as cell speed, directionality, mean square displacement, and directional persistence.