Cancer cell motility and invasion are critical targets for anticancer therapeutics. Whereas in vitro models could be designed for rapid screening with a view to investigate these targets, careful consideration must be given to the construction of appropriate model systems. Most investigations focus on two-dimensional (2-D) assays despite the fact that increasing evidence suggests that migration across rigid and planar substrates fails to recapitulate in vivo behavior. In contrast, few systems enable three-dimensional (3-D) cell migration to be quantitatively analyzed. We previously developed a digital holographic microscope (DHM) working in transmission with a partially spatial coherence source. This configuration avoids the noise artifacts of laser illumination and makes possible the direct recording of information on the 3-D structure of samples consisting of multiple objects embedded in scattering media, such as cell cultures in matrix gels. The software driving our DHM system is equipped with a time-lapse ability that enables the 3-D trajectories of living cells to be reconstituted and quantitatively analyzed.