Inhibins are glycoprotein hormones, composed of an α-subunit and either a βA or a βB subunit. They play a major role in the hypothalamus-pituitary-gonad feedback system to negatively regulate FSH secretion, thereby regulating spermatogenesis and folliculogenesis. Total inhibin is a useful detection marker for some early-stage ovarian cancers; in particular, it is an excellent biomarker for granulosa cell tumours (GCT). GCTs have a tendency for late recurrence, often many years after initial diagnosis. Thus, measuring inhibin is useful for long term monitoring for recurrent GCT. Currently, an efficient and cost-effective total inhibin assay is not available, highlighting a need to develop a high-throughput total inhibin assay. Introduction of mass spectrometry in clinical endocrinology has played a tremendous role in improving the management of numerous endocrine diseases. Our aim is to establish a methodology using selective reaction monitoring (SRM)-based targeted proteomics using liquid chromatography-mass spectrometry (LC-MS) to detect total inhibin with enhanced sensitivity in serum. Using conditioned cell culture media from HEK293 cells overexpressing inhibin B, we successfully detected inhibin α peptides using a NanoLC/Q Exactive Orbitrap mass spectrometer. We observed that an inhibin a peptide sequence (STPLMSWPWSPSALR) was one of the top 8 detectable proteins in the conditioned media. Using the MASCOT analysis software, this peptide (observed M.W. of 1714.85) had a significant MASCOT score (74.26) with a retention time of 2142.1156 seconds corresponding to the MS/MS spectrum. These parameters are now being used in conjunction with a suitable enrichment of low abundant proteins in serum, in order to detect this inhibin α peptide in serum from GCT patients. The implications of this study will allow women with ovarian cancer to gain access to a highly specific and sensitive diagnostic assay for α-inhibin, subsequently predicting relapse and/or monitoring recurrence to achieve better overall survival rate.