Levels of the peptide hormone hepcidin negatively correlate with systemic iron status and are increased in disorders in which iron metabolism is secondarily disregulated, such as the anemia of chronic disease. Consequently, the ability to measure hepcidin in the clinical setting may have diagnostic value for a broad range of indications. We describe a novel quantitative matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry assay for hepcidin in human urine which involves (i) direct enrichment from minute volumes (5 microL) of minimally treated urine on the surface of a functionalized chip, (ii) quantification by the use of a stable isotope labeled internal standard, and (iii) analysis by MALDI-TOF. Performance features include a wide linear range (1-1000 nM; LOQ 2.5 nM), high accuracy (90-110% recovery) and precision (intraday CV 12.11%; interday CV 13.21%), and a strong correlation upon interlaboratory cross validation with an existing immunoassay. The assay is simple, accurate, and efficient, and the high-throughput performance features of the assay make large-scale clinical research studies feasible.