Enriched stable iron isotopes in combination with isotope pattern deconvolution and ICP-MS have been used to study the absorption and bioavailability of iron from supplemented formula milk administrated to lactating rats. The use of two enriched stable isotope tracers, one as the metabolic tracer (here 57Fe) and the other (54Fe) as quantitation tracer, is shown to provide quantitative data about endogenous and exogenous (supplemented) total Fe distribution in rat feces, urine, red blood cells (RBCs), serum, liver, and kidney. The proposed analytical methodology was validated using reference materials (serum, urine, and liver) spiked with both 54Fe and 57Fe. Quantitative information about iron absorption/bioavailability and/or metabolism can be obtained from the amounts of endogenous and exogenous iron found in the tissues and fluids analyzed, and about its kinetic after 2 weeks of iron supplementation. The obtained results are discussed in terms of iron exchanged and its half-life in lactating rats and the observed iron levels in serum, RBCs, liver, and kidney comparing nonsupplemented rats and maternal feed rats.