Characterization of glycosaminoglycans poses a challenge for current analytical techniques, as they are highly acidic, polydisperse and heterogeneous compounds. The purpose of this study is the separation and analysis of a partially depolymerized heparin‐like glycosaminoglycan by on‐line ion‐pairing reversed‐phase high‐performance liquid chromatography/electrospray mass spectrometry. The gas‐phase behavior of two synthesized glycosaminoglycans has been investigated. Dibutylamine was found to be the best suited ion‐pairing reagents for mass spectrometry analysis. The optimized ion‐pairing conditions provide reproducible and easily interpretable electrospray mass spectra in both negative and positive ESI modes. The glycosaminoglycans are detected as a non‐covalent complex with amines. In fact, the observed ionic species and their gas‐phase dissociation under CID conditions revealed the presence of salt bridge interactions in the gas phase. Copyright © 2011 John Wiley & Sons, Ltd.