Evolutionarily, iron-sulfur (Fe-S) proteins have been vital for cellular functioning and the persistence of life. These Fe-S proteins typically consist of 2Fe-2S or 4Fe-4S clusters, which perform roles such as electron transfer and catalysis. Processes such as metabolism, respiration, and DNA synthesis rely on these proteins to drive their progress. The homeostasis of Fe-S proteins and their clusters impacts the growth and overall health of most organisms. Consequently, defects in these proteins result in detrimental diseases such as anemia, ataxia, and myopathy. Similar diseases arise when defects exist in the biogenesis machinery of the Fe-S clusters themselves. However, we currently cannot use imaging to assess the Fe-S cluster levels on these proteins within animals. My thesis research aims to develop new tools for imaging Fe-S proteins by using fragmented fluorescent proteins with excitation and emission in the near-infrared region of the spectrum. Using human glutaredoxin 2 (GRX2), I created a dozen biosensors by fusing fragments of a split near-infrared fluorescent protein (IFP) to this dimerizing dithiol glutaredoxin. My results indicate that Escherichia coli expressing IFP fragments fused to GRX2 display significantly higher fluorescence than cells expressing the same IFP fragments fused to a C37A mutant of GRX2, which lacks the active site cysteine necessary for coordination to a 2Fe2S cluster and dimerization. Endpoint assays were used to show the differences in biosensor behavior at two different temperatures. The biosensors behave differently at 37°C and 25°C. At reduced temperatures, the C37A mutant of GRX2 fluoresce to a greater extent, which may arise from dimerization without binding to a 2Fe2S cluster. In addition, I showed that dynamic measurements with these biosensors display differences in signal over time, and these assays could be easily used to analyze the behavior of GRX2 in different cysteine desulfurase knockout strains. The split IFP fused to GRX2 will be applicable when investigating 2Fe2S cluster availability in various microorganisms and multicellular organisms. The results of my thesis work have yielded a new imaging tool that can be used to advance the understanding of how genetics and environment alter the construction of 2Fe2S clusters and affect dimerization of Fe-S proteins. Finally, comparisons of biosensor expression will enhance the understanding of disease states due to defects in mutations in Fe-S proteins or the Fe-S biogenesis machinery.