Hepcidin has been identified as the principle regulatory hormone essential for iron homeostasis. Quantitative analysis of hepcidin in bodily fluids provides an insight into the pathogenesis of disorders of iron metabolism such as hereditary hemochromatosis and anemia of chronic disease. This study describes the use of solid phase extraction (SPE) as a preparative step followed by matrix assisted laser desorption/ionization-orthogonal-time-of-flight mass spectrometry (MALDI-TOF MS) with internal standard for the quantitative analysis of unlabelled urinary hepcidin. More than 70% extraction recovery of hepcidin (hepcidin-25) with monoisotopic resolution was achieved. Urinary creatinine was analyzed using HPLC-UV/Vis with hepcidin-25 levels of 2.2 to 2.7 nmol/mmol of creatinine observed in healthy controls. Spot-to-spot variation of hepcidin standard additions was less than 3.5%. Intra- and inter-day precision assay of less than 9.5% relative standard deviation was achieved with less than 0.5% variation between the intra-day assay data. In summary, a validated non-invasive method has been developed for the quantification of unlabelled urinary hepcidin-25 which can be used to screen for iron-related disorders such as hemochromatosis in iron-overloaded patients.