Ghrelin, a 28-amino acid peptide with an n-octanoyl modification indispensable for its biological activity, is synthesized principally in the stomach and released in response to acute and chronic energy imbalances. Due to increased interest in ghrelin measurement, a standardized method of sample collection is required. The present study sought to investigate the effect of a variety of anticoagulants and storage conditions on ghrelin stability. In whole blood and plasma, acylated ghrelin was found to be highly unstable, as ester bonding can be degraded both chemically and enzymatically under these conditions. To acquire accurate data on ghrelin levels, blood samples should be collected with ethylene diamine tetra-acetic acid-aprotinin and centrifuged within 30 minutes under cooled conditions. The stability of ghrelin gradually decreased in untreated plasma, with 40% degradation after 6 hours of storage at 37°C. Storage at 4°C and acidification of plasma to pH 3–4 maintained ghrelin stability. In acidified plasma (pH 4), ghrelin levels were not altered by up to 4 cycles of freezing and thawing. After intravenous administration to anesthetized rats, plasma ghrelin levels rapidly decreased with a half-life of 8 minutes. In conclusion, as ghrelin is highly unstable, it is necessary to standardize the preparation of samples to ensure reliable ghrelin measurements.