Iron in blood plasma is bound to its transport protein transferrin, which delivers iron to most tissues. In iron overload and certain pathological conditions, the carrying capacity of transferrin can become exceeded, giving rise to non-transferrin-bound iron, which is taken up preferentially by the liver, kidney, pancreas, and heart. The measurement of tissue transferrin- and non-transferrin-bound iron (TBI and NTBI, respectively) uptake in vivo can be achieved via intravenous administration of 59Fe-labeled TBI or NTBI followed by gamma counting of various organs. Here we describe a detailed protocol for the measurement of TBI and NTBI uptake by mouse tissues.