Hepcidin, a cysteine-rich peptide hormone, secreted mainly by the liver, plays a central role in iron metabolism regulation. Emerging evidence suggests that disordered iron metabolism is a risk factor for various types of diseases including cancers. However, it remains challenging to apply current mass spectrometry (MS)-based hepcidin assays for precise quantification due to the low fragmentation efficiency of intact hepcidin as well as synthesis difficulties for the intact hepcidin standard. To address these issues we recently developed a reliable sensitive targeted MS assay for hepcidin quantification from clinical samples that uses fully alkylated rather than intact hepcidin as the internal standard. Limits of detection and quantification were determined to be <0.5 ng/mL and 1 ng/mL, respectively. Application of the alkylated hepcidin assay to 70 clinical plasma samples (42 non-cancerous and 28 ovarian cancer patient samples) enabled reliable detection of endogenous hepcidin from the plasma samples, as well as conditioned culture media. The hepcidin concentrations ranged from 0.0 to 95.6 ng/mL across non-cancerous and cancer plasma specimens. Interestingly, cancer patients were found to have significantly higher hepcidin concentrations compared to non-cancerous patients (mean: 20.6 ng/ml for cancer; 5.94 ng/ml for non-cancerous) (p value < 0.001). Our results represent the first application of the alkylated hepcidin assay to clinical samples and demonstrate that the developed assay has better sensitivity and quantification accuracy than current MS-based hepcidin assays without the challenges in synthesis of intact hepcidin standard and accurately determining its absolute amount.