Hepcidin is a recently recognized defensin-like peptide, which is considered to be the central regulator of iron metabolism. Hepcidin decreases the expression of iron transporting molecules. Hepcidin reduces gastrointestinal iron absorption, iron release from the macrophages, and hence it decreases serum iron levels. Clarification of hepcidin role in iron homeostasis could provide an explanation to anemia of inflammation and chronic diseases. At start of our work there was no commercially available method for measuring urine hepcidin levels. The aim of our study was to develop an easily achievable, reliable quantification method for the determination of urine hepcidin levels in human, in addition to examine a possible association of hepcidin with neonatal iron homeostasis. According to the sequence of native, human hepcidin we have synthesized peptide derivatives from which 1-7 peptide derivatives might be suitable representatives of the 25-amino-acid form of hepcidin in immune adsorption method. We presented a novel laser-desorption mass spectrometry based semi-quantitative, reproducible method for measuring hepcidin concentration in human urine first using the synthesized peptide derivative acetyl-1-25 peptide as hepcidin related internal standard. We described an easy and quickly achievable solid-phase extraction method which is suitable for purification of urine and concentration of hepcidin. In our study we have first measured serum prohepcidin and urine hepcidin in healthy human newborns. Serum prohepcidin levels showed no significant changes, however, urine hepcidin levels increased significantly during the first postnatal days. Serum prohepcidin and urine hepcidin levels showed no significant association in healthy human newborns. Associations have been demonstrated between cord blood prohepcidin values and mean corpuscular hemoglobin concentration as well as between urine hepcidin levels and serum iron and total iron binding capacity values. We have demonstrated that neonates with detectable non-protein-bound iron levels in cord blood were presented with lower prohepcidin concentrations. In summary, our results suggest a possible link between hepcidin and early iron adaptation of newborn's, however, further investigations should be done to elucidate this issue.