AbstractAn array of NMR spectroscopy experiments have been carried out to obtain conformationally dependent 1H,13C‐ and 13C,13C‐spin–spin coupling constants in the trisaccharide α‐L‐Rhap‐(1 → 2)[α‐L‐Rhap‐(1 → 3)]‐α‐L‐Rhap‐OMe. The trisaccharide was synthesized with 13C site‐specific labeling at C2′ and C2″, i.e. in the rhamnosyl groups in order to alleviate 1H spectral overlap. This facilitated the measurement of a key trans‐glycosidic proton–proton cross‐relaxation rate using 1D 1H,1H‐T‐ROESY experiments as well as a 3JC, H coupling employing 1D 1H,13C‐long‐range experiments, devoid of potential interference from additional J coupling. By means of both the natural abundance compound and the 13C‐labeled sample 2D 1H,13C‐J‐HMBC and 1H,13C‐HSQC‐HECADE NMR experiments, total line‐shape analysis of 1H NMR spectra and 1D 13C NMR experiments were employed to extract 3JC, H, 2JC, H, 3JC, C, and 1JC, C coupling constants. The 13C site‐specific labeling facilitates straightforward determination of nJC, C as the splitting of the 13C natural abundance resonances. This study resulted in eight conformationally dependent coupling constants for the trisaccharide and illustrates the use of 13C site‐specific labeling as a valuable approach that extends the 1D and 2D NMR methods in current use to attain both hetero‐ and homonuclear spin–spin coupling constants that subsequently can be utilized for conformational analysis. Copyright © 2011 John Wiley & Sons, Ltd.