[FeFe] hydrogenases catalyze proton reduction and hydrogen oxidation with high rates and efficiency under physiological conditions, but are highly oxygen sensitive. The [FeFe] hydrogenase from Desulfovibrio desulfuricans (DdHydAB) can be purified under air in an oxygen stable inactive state H-ox(air). The formation of the H-ox(air) state in vitro allows the handling of hydrogenases in air, making their implementation in biotechnological applications more feasible. Here, we report a simple and robust protocol for the formation of the H-ox(air) state in DdHydAB and the [FeFe] hydrogenase from Chlamydomonas reinhardtii, which is based on high potential inactivation in the presence of sulfide.