Hydrogenases catalyze the reversible formation of H2, and they are key enzymes in the biological cycling of H2. H isotopes have the potential to be a very useful tool in quantifying hydrogen ion trafficking in biological H2 production processes, but there are several obstacles that have thus far limited the application of this tool. Here, we describe a new method that overcomes some of these barriers and is specifically designed to measure isotopic fractionation during enzyme-catalyzed H2 evolution. A key feature of this technique is that purified hydrogenases are employed, allowing precise control over the reaction conditions and therefore a high level of precision. In addition, a custom-designed high-throughput gas chromatograph/isotope ratio mass spectrometer is employed to measure the isotope ratio of the H2. Using our new approach, we determined that the fractionation factor for H2 production by the [NiFe]-hydrogenase from Desulfovibrio fructosovorans is 0.273 ± 0.006. This result indicates that, as expected, protons are highly favored over deuterium ions during H2 evolution. Potential applications of this newly developed method are discussed. Copyright © 2011 John Wiley & Sons, Ltd.