Lipoyl synthase (LipA in bacteria) is a radical S-adenosylmethionine (SAM) enzyme that catalyzes the second step of the de novo biosynthesis of the lipoyl cofactor: the insertion of sulfur at C6 and C8 of a pendant octanoyl chain. In addition to the [4Fe–4S] cluster that is characteristic of the radical SAM (RS) enzymes, LipA contains a second [4Fe–4S] cluster that, though controversial, has been proposed to be degraded during turnover to supply the inserted sulfur atoms. A consequence of this proposed role is that the destruction of its iron–sulfur cluster renders the enzyme in an inactive state. Recently, it was shown that Escherichia coli proteins NfuA or IscU can confer catalytic properties to E. coli LipA in vitro. In this chapter, we present methods for characterizing LipA and analyzing its activity in vitro, and provide strategies to monitor the pathway for the regeneration of LipA's auxiliary cluster by E. coli iron–sulfur carrier protein NfuA.