Hydrogenases control the proton concentration in cells, which is an essential function for hydrogen metabolism in several microorganisms. Some [NiFe]-hydrogenases are catalytically active under air and are thus of great interest for developing bio-inspired synthetic models and new devices for clean energy conversion. Here, we provide an overview of the structural basis of the reaction mechanism of [NiFe]-hydrogenases, and the recent development of a new assay method which may uncover hidden properties of hydrogenases.