Biotin synthase (BioB) catalyzes the oxidative insertion of a sulfur atom between the C6 methylene and the C9 methyl positions in dethiobiotin. The enzyme couples oxidation of each carbon position to reduction of the S-adenosyl-l-methionine (SAM) sulfonium center, generating 5'-deoxyadenosine and l-methionine, products that are characteristic of enzymes from the radical SAM superfamily. In bacteria, biotin biosynthesis is tightly regulated by the dual-function BirA repressor/holocarboxylase synthetase, resulting in very low levels of all biotin biosynthetic enzymes such that activity-based purification of BioB from the native organism is virtually impossible. However, overexpression and purification of recombinant BioB from E. coli are straight forward and, in contrast with many radical SAM enzymes, can be carried out under aerobic conditions. The active enzyme contains two iron-sulfur clusters, and the characterization and manipulation of these clusters are essential for a thorough understanding of enzyme catalysis and stability. An optimized in vitro assay for BioB is described herein that requires use of an auxiliary protein reducing system and must be carried out under anaerobic conditions to prevent oxidative damage to the reduced iron-sulfur clusters. Three methods for detection of biotin are described, with discussion of the advantages and limitations of each method. Challenges that may be encountered in adapting these assays to other organisms are also discussed.