To date, most of the studies in the field of cell migration have been applied to two-dimensional (2D) models. To mimic the three-dimensional (3D) conditions similar to those observed in vivo during tumor invasion, we developed a 3D model of cell migration in which cells were embedded in a collagen I matrix placed in a double-compartment chamber. Using time-lapse videomicroscopy and interactive cell tracking in a four-dimensional data set, we determined the cell trajectories and their migration kinetics. We compared the 2D and 3D migratory behavior of a noninvasive cell line (16HBE) with the migratory behavior of an invasive cell line (BZR). Our results show that the 3D migration kinetics of the noninvasive cell line were lower than the migration kinetics of the invasive cell line. In contrast, in 2D models, no significant difference was observed between the two cell lines. To validate our 3D model, we further investigated the effect of epidermal growth factor (EGF), a promoter of tumor cell motility and invasion on the noninvasive cell line (16HBE). EGF increased significantly the migration kinetics of the noninvasive cell line. Our results show that the 3D model of cell migration allowed us to differentiate the migratory behavior of invasive and noninvasive cells and that such a model can help in the development of molecular targeted therapy as it approaches the in vivo conditions.