Seeding a monolayer of primary human endothelial cells on the surface of a polymerized three-dimensional collagen matrix in the presence of pro-angiogenic stimuli allows manipulation and analysis of rapid sprouting responses. This protocol is useful for elucidating incompletely defined intracellular mechanisms downstream of pro-angiogenic factors that regulate sprout formation and initiation, and can also be used to test the efficacy of pro-and anti-angiogenic compounds. We present protocols to culture endothelial cells, prepare three-dimensional collagen matrices and quantify and image rapid endothelial sprouting responses (24 h). This protocol can be carried out using either type I or type II collagen matrices with primary endothelial cells isolated from macrovascular and microvascular sources of varying species.