Background: The peptide hormone hepcidin-25 plays an important role in iron metabolism. Low or high levels of hepcidin-25 are associated with various iron disorders; therefore, hepcidin-25 is an important biomarker. This study describes an easy and fast analytical assay for the quantification of hepcidin-25 with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Methods: Sample preparation was performed by protein precipitation with trichloroacetic acid, and injection onto a LC-MS/MS was directly conducted from a LoBind 96-well plate. Results: The concentration range covered by the quality control samples, ranged from 0.25 nmol/L (12.3% CV) to 11.9 nmol/L (CV < 9%). Matrix effect was limited (mean recovery of 99.9% with a CV of 6.4%). The assay was validated for serum, EDTA and heparin plasma. An international secondary reference material was used for calibration. The reference interval (90% CL) was estimated for hepcidin-25 by analysing serum and plasma samples from 156 healthy subjects with a lower limit: 0.12 (0.07–0.19) and upper limit: 11.2 nmol/L (9.5–13.0). Conclusions: We present a fast and easy assay for the quantification of hepcidin-25 in serum and plasma samples. The assay was successfully used for the detection of various forms of hereditary haemolytic anaemias, to characterize the interplay between erythropoiesis and iron levels.