Auxin, a major phytohormone, plays a pivotal role throughout the plant life cycle. While indole 3-acetic acid (IAA) is the major natural auxin found in plants, there are other natural and many synthetic chemicals with auxin activity. Ability to test the auxin activity in a simple cell-free assay will facilitate the identification of different chemicals with auxin activity. Moreover, the same assay can be used to identify the chemicals with antiauxin activity. Here we describe an in vitro pull-down assay that can be easily used to test auxin activity of different chemicals. Further, the concept of this assay can be used to test the activity of other hormones with similar mode of action or small ligand-based protein-protein interactions.