Finding the targets of natural products is of key importance in both chemical biology and drug discovery, and deconvolution of cofactor interactomes contributes to the functional annotation of the proteome. Identifying the proteins that underlie natural compound activity in phenotypic screens helps to validate the respective targets and, potentially, expand the druggable proteome. Here, we present a generally applicable protocol for the photoactivated immobilization of unmodified and microgram quantities of natural products on diazirine-decorated beads and their use for systematic affinity-based proteome profiling. We show that among 31 molecules of very diverse reported activity and biosynthetic origin, 25 could indeed be immobilized. Dose-response competition binding experiments using lysates of human or bacterial cells followed by quantitative mass spectrometry recapitulated targets of 9 molecules with <100 μM affinity. Among them, immobilization of coenzyme A produced a tool to interrogate proteins containing a HotDog domain. Surprisingly, immobilization of the cofactor flavin adenine dinucleotide (FAD) led to the identification of nanomolar interactions with dozens of RNA-binding proteins.