Sulfur trafficking systems are multiprotein systems that synthesize sulfur-containing cofactors such as iron-sulfur clusters. The sulfur is derived enzymatically from cysteine and transferred between nucleophilic cysteine residues within proteins until incorporation into the relevant cofactor. As these systems are poorly understood, we have developed an in vivo method for characterizing these interactions and have applied our method to the SUF system of Escherichia coli, which is responsible for iron-sulfur cluster biogenesis under oxidative stress and iron limitation. Proteins that interact covalently with SufE were trapped in vivo, purified, and identified by mass spectrometry. We identified SufE-SufS and SufE-SufB interactions, interactions previously demonstrated in vitro, indicating that our method has the ability to identify physiologically relevant interactions. The sulfur acceptor function of SufE is likely due to the low pK(a) of its active site C51, which we determined to be 6.3 ± 0.7. We found that SufE interacts with several Fe-S cluster proteins, further supporting the validity of the method, and with tryptophanase, glutaredoxin-3, and glutaredoxin-4, possibly suggesting a role for these enzymes in iron-sulfur biogenesis by the SUF system. Our results indicate that this method could serve as a general tool for the determination of sulfur trafficking mechanisms.