Complete 1H and 13C resonance assignments were made for a new type of 3 beta,7 beta-dihydroxy-5-cholen-24-oic acid doubly conjugated with sulfuric acid at C-3 and N-acetylglucosamine at C-7 and its glycine- and taurine-amidated triple-conjugates by the combined use of several homonuclear and heteronuclear shift-correlated 2D NMR techniques. The effects of sulfation at C-3, N-acetylglucosaminidation at C-7, and aminoacyl amidation at C-24 on the 1H and 13C chemical shifts and signal multiplicity were clarified. The shielding data serving to characterize each of the bile acid multi-conjugates are also discussed.