The extracellular matrix (ECM) is a network of molecules that provide a structural framework for cells and tissues and helps facilitate intercellular communication. Three-dimensional cell culture techniques have been developed to more accurately model this extracellular environment for in vitro study. While many cell processes during migration through 3D matrices are similar to those required for movement across rigid 2D surfaces, including adherence, migration through ECM also requires cells to modulate and invade this polymeric-mesh of ECM. In this video, we will present the structure and function of ECM and the basic mechanisms of how cells migrate through it. Then, we will examine the protocol of an assay for tube formation by endothelial cells, whose steps can be generalized to other experiments based on 3D matrices. We will finish by exploring several other biological questions that can be addressed using ECM invasion assays.