In this study, a selective and sensitive new 2D-LC method was developed for the determination of ramelteon in human serum. Separations were performed using the following systems: the first dimension consisted of a C18 (50 mm x 2.1 mm) pre-separation column and the mobile phase of 45% acetonitrile: 55% 10 mM ortho-phosphoric acid solution mixture and the second dimension consisted of a pentafluorophenyl propyl (150 mm x 4.6 mm) separating column and the mobile phase of 40% methanol: 60% 2.25 mM ortho-phosphoric acid solution mixture. Two systems were connected with a 6 port valve including 200 mu L sample loop and separations were performed continuously through injections. Detection wavelength has been selected as 203 nm for both systems. Extraction of drug substance from serum samples was performed using protein precipitation method with methanol and the calibration curve was linear over a concentration range of 1-50 ng/mL. The accuracy and precision of the method were determined as 1.49-4.24% and 1.75-3.63% ranges for RSD and RME, respectively. The absolute recovery of the drug was found as 80.28%. The developed method was successfully applied for the determination of ramelteon in serum samples collected from a volunteer who had received 8 mg ramelteon orally. (C) 2015 The Authors. Published by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).