Iron–sulfur (Fe/S) proteins play crucial roles in living cells by participating in enzyme catalysis, electron transfer and the regulation of gene expression. The biosynthesis of the inorganic Fe/S centers and their insertion into apoproteins require complex cellular machinery located in the mitochondria (Fe/S cluster (ISC) assembly machinery systems) and cytosol (cytosolic Fe/S protein assembly (CIA) systems). Functional defects in Fe/S proteins or their biogenesis components lead to human diseases underscoring the functional importance of these inorganic cofactors for life. In this protocol, we describe currently available methods to follow the activity and de novo biogenesis of Fe/S proteins in eukaryotic cells. The assay systems are useful to follow Fe/S protein maturation in different cellular compartments, identify novel Fe/S proteins and their biogenesis factors, investigate the molecular mechanisms underlying the maturation process in vivo and analyze the effects of genetic mutations in Fe/S protein-related genes. Comprehensive analysis of one biogenesis component or target Fe/S protein takes about 10 d.