Appetite-stimulating peptide hormone Ghrelin exists in both Active acylated [octanoyl group on serine-3] and Inactive unacylated forms. Metabolic effects of Ghrelin are mediated by Active Ghrelin. Ratios of Total and Active Ghrelin are measured as biomarkers in a variety of studies. In our quest for a reliable kit for quantification of Ghrelin, we evaluated two commercial kits from the same vendor. Though the kit for Active Ghrelin worked well under assay conditions specified by the kit manufacturer, significant changes in incubation conditions were required to achieve optimum sensitivity of the ELISA kit for Total Ghrelin. In this case study, a fit-for purpose modification of the Total Ghrelin assay is described and analytical method qualification characteristics of the assay are presented.