Hepcidin, a key regulator of iron homeostasis, is known to have three isoforms: hepcidin-20, -22, and -25. Hepcidin-25 is thought to be the major isoform and the only one known to be involved in iron metabolism; the physiological roles of other isoforms are poorly understood. Because of its involvement in the pathophysiology of hereditary hemochromatosis and the anemia of chronic disease, the regulatory mechanisms of hepcidin expression have been extensively investigated, but most studies have been performed only at the transcriptional level. Difficulty in detecting hepcidin has impeded in vitro research. In the present study, we developed a novel method for simultaneous quantification of hepcidin-20, -22, and -25 in the media from hepatoma-derived cell lines. Using this method, we determined the expression patterns of hepcidin isoforms and the patterns of responses to various stimuli in human hepatoma-derived cultured cells. We found substantial differences among cell lines. In conclusion, a novel method for simultaneous quantification of hepcidin isoforms is presented. Heterogeneous expressions of hepcidin isoforms in human hepatoma-derived cells were revealed by this method. We believe our method will facilitate quantitative investigation of the role hepcidin plays in iron homeostasis.