Fluorescent microspheres are commonly used to assess bone blood supply in large animals, but the technique is not widely used in smaller mammals, as traditional methods such as reference blood sampling, ventilation and catheterization are not easily applied. This protocol describes a viable alternative for measuring bone and organ perfusion in mice using modified fluorescent microsphere techniques. Microspheres are injected directly into the left heart and a reference tissue is used to calculate relative bone and organ blood supply. On the basis of a sample of 15 mice with 5 tissues each, the entire protocol takes 140.5 h to complete from animal preparation through statistical analysis. This timing includes 72 h of mandated pauses for bone decalcification and digestion, as well as 48 h for data analysis. Exclusive of pauses or additional analyses that could increase the time required, this protocol takes 20.5 h bench time to complete.