The objective of this study was to evaluate and compare different sample treatments and storage conditions in order to determine optimal conditions for total and acylated canine ghrelin measurement using ELISA assays. Under the conditions assessed in this study, total ghrelin concentration was stable when stored at room temperature or under cooled conditions (4°C) for approximately six hours regardless of the type of anticoagulant used. When samples were stored at under −20°C, three freeze/thaw cycles during storage did not alter total ghrelin concentration if serum or plasma treated with EDTA or with EDTA-aprotinin is used. When samples were stored at under −80°C, no significant differences were observed after three freeze/thaw cycles in total ghrelin concentrations in serum or plasma treated with EDTA, EDTA-aprotinin or heparin. While acylated ghrelin showed to be very instable, its analysis should be performed within an hour of sample collection or stored at −80°C preferably in serum in order to acquire accurate data. Addition of HCl did not improve total or acylated ghrelin stability. Furthermore, acidification of plasma samples decreased the stability of total ghrelin and impeded determination of acylated ghrelin with the ELISA used in this study.