MitoNEET is an integral protein of the outer mitochondrial membrane and is the flagship of a small family of proteins whose hallmark is the presence of a CDGSH domain. Initially annotated as a zinc finger, the CDGSH domain actually binds a redox-active 2Fe-2S cluster, giving mitoNEET the distinction of being the first 2Fe-2S protein identified in the outer membrane of mitochondria. This chapter describes methods for isolating mitochondrial membrane fractions that are enriched in mitoNEET, generating constructs for the expression of recombinant mitoNEET protein and analyzing the 2Fe-2S cluster of mitoNEET in vitro.