Cell invasion requires that cells navigate complex three-dimensional matrices in vivo. Topological cues are provided and three-dimensional cell migration and invasion facilitated by the alignment of collagen fibers proximal to tumors. In order to better understand the molecular mechanisms by which cells recognize and migrate along aligned matrices, in vitro assays are needed that recapitulate topological features of the in vivo matrix. Here, we describe two approaches for creating aligned three-dimensional collagen matrices, both dependent and independent of cell-mediated alignment. Approaches to quantify alignment and visualize the collagen matrix relative to the cells by second-harmonic generation are included. These assays are readily adaptable to a variety of cells and biological questions related to three-dimensional cell migration.