In vitro models are useful for dissecting cell behavior under controlled conditions. Angiogenesis is a multistep process where endothelial cells (ECs) are activated by pro-angiogenic factors to degrade the basement membrane, migrate into the surrounding matrix, and form sprouting structures connecting neighboring vessels. Sphingosine-1-phosphate (S1P), a biologically active sphingolipid, promotes vessel morphogenesis and angiogenesis during embryonic development and in adults under normal and pathological conditions via its actions on ECs. Here, we describe an in vitro endothelial morphogenic assay that is significantly enhanced by S1P. This method allows for testing whether molecules and their related signaling pathways regulate the initiation of angiogenic sprouts stimulated by S1P, as well as whether individual compounds have pro- or anti-angiogenic properties.