Cysteine desulfurase plays central role in mitochondrial iron-sulfur cluster biogenesis not only by providing sulfur through the catalysis of L-cysteine to L-alanine but also by serving as the platform for the assembly of other components of the biosynthetic machinery, including ISCU, frataxin, and ferredoxin. Human mitochondrial cysteine desulfurase complex consists of a homodimer with three components: NFS1, ISD11 and acyl carrier protein (ACP). In this study we used chemical crosslinking coupled with tandem mass spectrometry (XC-MS) to investigate the structures of cysteine desulfurase complexes.