This paper describes a screening assay, compatible with high-throughput bioprospecting or molecular biology methods, for assessing biological hydrogen (H2) production. While the assay is adaptable to various physical configurations, we describe its use in a 96-well, microtiter plate format with a lower plate containing H2-producing cyanobacteria strains and controls and an upper, membrane-bottom plate containing a color indicator and a catalyst. H2 produced by cells in the lower plate diffuses through the membrane into the upper plate, causing a color change that can be quantified with a microplate reader. The assay is reproducible, semiquantitative, sensitive down to at least 20 nmol of H2, and largely unaffected by oxygen, carbon dioxide, or volatile fatty acids at levels appropriate to biological systems.