AbstractBackgroundMost phenomena in developmental biology involve or depend upon cell migration. This article describes a comprehensive framework for the characterization and analysis of trajectories defined by cell movement. The following two perspectives are considered: (a) the behavior of each individual cell and (b) interactions between neighboring pairs of cells.MethodsThe measurements considered for individual trajectories include the velocity magnitude and orientation, maximum spatial dispersion, displacement effectiveness, and displacement entropies. Interactions between two trajectories are characterized by comparing the respective velocities.ResultsThe potential of the overall framework is illustrated using data of moving cells in different biological environments. The work shows that it is possible to use the new algorithm presented here to characterize cell motility.ConclusionsThe features of the algorithm were successful in determining the motility changes under different experimental conditions. © 2005 Wiley‐Liss, Inc.