We describe here how to prepare and analyze serum-free cultures of mouse aortic rings for the study of angiogenesis. Removal of serum from the culture medium helps visualize angiogenic neovessels by reducing fibroblast outgrowth and allows evaluation of test substances without interference by serum molecules. Mouse aortic rings are, however, unable to sprout using the serum-free method originally developed for the rat aortic ring assay, unless stimulated with exogenous angiogenic factors. This limitation is due to the small size of the mouse aortic rings and can be overcome by miniaturizing the assay. Volume reduction of culture medium concentrates endogenously released factors enabling angiogenic sprouting. Using this modified approach, mouse aortic rings embedded in thin collagen gels and cultured in 1/10th the volume of serum-free medium used for rat aortic rings spontaneously generate pericyte-coated endothelial sprouts, which can be further stimulated with exogenous angiogenic factors. This method can be used to study the activity of angiogenic regulators and the angiogenic response of genetically modified mice.