Chemical biology approaches have been instrumental in understanding the mode of action of brassinosteroids, a group of plant steroid hormones essential for plant development and growth. The small molecules used for such approaches include inhibitors of biosynthetic enzymes and signaling components. Additionally, recent structural data on the brassinosteroid receptor complex together with its ligand brassinolide, the most active brassinosteroid, and knowledge on its different analogs have given us a better view on the recognition of the hormone and signaling initiation. Moreover, a fluorescently labeled brassinosteroid enabled the visualization of the receptor ligand pair in the cell. Given the insights obtained, small molecules will continue to provide new opportunities for probing brassinosteroid biosynthesis and for unraveling the dynamic and highly interconnected signaling.