Abstract Background Hepcidin is a 25-residue peptide hormone crucial to iron homeostasis. It is essential to measure the concentration of hepcidin in cells, tissues and body fluids to understand its mechanisms and roles in physiology and pathophysiology. With a mass of 2791 Da hepcidin is readily detectable by mass spectrometry and LC-ESI, MALDI and SELDI have been used to estimate systemic hepcidin concentrations by analysing serum or urine. However, peak heights in mass spectra may not always reflect concentrations in samples due to competition during binding steps and variations in ionisation efficiency. Thus the purpose of this study was to develop a robust assay for measuring hepcidin using a stable isotope labelled hepcidin spiking approach in conjunction with SELDI-TOF-MS. Results We synthesised and re-folded hepcidin labelled with ¹³C/¹⁵N phenylalanine at position 9 to generate an internal standard for mass spectrometry experiments. This labelled hepcidin is 10 Daltons heavier than the endogenous peptides and does not overlap with the isotopic envelope of the endogenous hepcidin or other common peaks in human serum or urine mass spectra and can be distinguished in low resolution mass spectrometers. We report the validation of adding labelled hepcidin into serum followed by SELDI analysis to generate an improved assay for hepcidin. Conclusion We demonstrate that without utilising a spiking approach the hepcidin peak height in SELDI spectra gives a good indication of hepcidin concentration. However, a stable isotope labelled hepcidin spiking approach provides a more robust assay, measures the absolute concentration of hepcidin and should facilitate inter-laboratory hepcidin comparisons.