Hepcidin-25 is a peptide-hormone that has been proposed as the key biomarker for the diagnosis and monitoring of iron disorders. Structurally, hepcidin-25 is a S-rich peptide (with 8 cysteines and 1 methionine) that contains a metal binding motif in the N-terminus. That domain binds preferably Cu(II) ion forming a stable complex. Such selective binding can be used as mean to determine hepcidin-25 in biological fluids by highly sensitive Cu measurement. Thus, we use liquid chromatography coupled to inductively coupled plasma mass spectrometry (LC-ICP-MS) to perform hepcidin-25 determination via Cu detection. For this purpose, the incubation conditions were optimized to address the complex formation and stability by electrospray-MS (ESI-q-TOF). It was found that Cu:hepcidin-25 complex is stable under physiological conditions and shows an equimolar stoichiometry (1:1). The collisional induced dissociation (CID) experiments confirmed the specific binding of Cu to the N-terminal motif. For Cu quantification, two isotope dilution strategies have been developed. The first one, including postcolumn addition of a (65)Cu spike and the second, by synthesizing the labeled (65)Cu:hepcidin-25 complex as tracer (species-specific). Both methods have been optimized and critically compared in real samples. The determination of hepcidin-25 in different serum samples from healthy individuals based on Cu monitoring showed a mean value of 21.6 ng mL(-1) which is in good agreement to previously published data.