A simple pretreatment method and separation mode for the LC-ESI-MS/MS determination of adenosine in human plasma have been developed. Deproteinization by acetonitrile and ultrafiltration followed by chromatographic separation with a hydrophilic interaction chromatographic (HILIC) column give a highly sensitive MS/MS response without ionic suppression caused by the matrix compounds in human plasma. In addition, the presence of ammonium acetate in the mobile phase contributes to high sensitivity in MS/MS detection, facilitating the ionization of adenosine. This method seems to be amenable to the treatment of many samples in clinical practice.