The current needs to understand gene function in plant biology increasingly require more dynamic and conditional approaches opposed to classic genetic strategies. Gene redundancy and lethality can substantially complicate research, which might be solved by applying a chemical genetics approach. Now understood as the study of small molecules and their effect on biological systems with subsequent target identification, chemical genetics is a fast developing field with a strong history in pharmaceutical research and drug discovery. In plant biology however, chemical genetics is still largely in the starting blocks, with most studies relying on forward genetics and phenotypic analysis for target identification, whereas studies including direct target identification are limited. Here, we provide an overview of recent advances in chemical genetics in plant biology with a focus on target identification. Furthermore, we discuss different strategies for direct target identification and the possibilities and challenges for plant biology.