Aerobic flavoenzymes are flavin-dependent enzymes that use oxygen to catalyze a diverse set of redox reactions, and they are essential to the proper function of biosynthetic secondary metabolism. Along with other enzymatic domains found in biosynthesis, flavoenzyme domains are attractive targets to investigate, owing to their role in processing substrates within modular synthases and with other protein domains. In this dissertation, a review of flavoenyzmes biosynthesis and applications in chemical biology is presented. Additionally, we report the successful development of mechanism-based inhibitors and probes of aerobic flavoenzyme activity.