Hydrogen bonds play critical roles in protein structure, stability, and function. Conventionally, hydrogen bonds are mainly determined by X-ray crystallography and NOE-based NMR spectroscopy in indirect manners. In recent years, it was demonstrated that hydrogen bonds can be directly detected through NMR measurements of trans-hydrogen-bond scalar coupling constants. Here we report across hydrogen-bond protium/deuterium isotope effects in a 35 kDA protein observed with the isotopomer-selective TROSY NMR technique (Liu at al. J. Biomol. NMR 2006, 36, 205-214; Liu at al. J. Magn. Reson. 2007, 186, 319-326) and show that such isotope effects can be used to detect a most common type of bifurcated hydrogen bonds, in which a heavy atom, usually oxygen, is involved in two hydrogen bonds, including a pair of bifurcated hydrogen bonds involving a bound water molecule.