RNA plays a central role in biological processes, and its activity is regulated by a host of diverse chemical and biochemical mechanisms including post-transcriptional modification and interactions with RNA-binding proteins. Here, we describe our efforts to illuminate RNA biology through the application of chemical tools, focusing on post-transcriptional regulatory mechanisms. We describe the development of an activity-based protein profiling approach for discovery and characterization of RNA-modifying enzymes. Next, we highlight novel approaches for RNA imaging based upon metabolic labeling with modified nucleosides and engineering of the nucleotide salvage pathway. Finally, we discuss profiling RNA-protein interactions using small molecule-dependent RNA editing and synthetic photo-cross-linkable oligonucleotide probes. Our work provides enabling technologies for deciphering the complexity of RNA and its diverse functions in biology.