Chemical biology is an interdisciplinary scientific field covering chemistry and biology. It uses chemical-based molecules to reveal or control biological processes. Due to the nature and potential of chemicals, they provide an alternative way to identify novel genetic elements associated with redundancy or lethality. In addition, bioactive chemicals can be applied practically as pharmaceutical drugs or agrochemicals. In plant science, many biologically active small molecules were isolated so far from high-throughput screenings using libraries of diverse compounds and their successes demonstrated the usefulness and advantage of this approach. The use, experimental design, tips and future perspective of chemical screening procedures are described herein by sharing experiences based on the previous screening campaigns.