Auxin modulates diverse plant developmental pathways through direct transcriptional regulation and cooperative signaling with other plant hormones. Genetic and biochemical approaches have clarified several aspects of the auxin-regulated networks; however, the mechanisms of perception and subsequent signaling events remain largely uncharacterized. To elucidate unidentified intermediates, we have developed a high-throughput screen for identifying small molecule inhibitors of auxin signaling in Arabidopsis. Analysis of 10,000 compounds revealed several potent lead structures that abrogate transcription of an auxin-inducible reporter gene. Three compounds were found to interfere with auxin-regulated proteolysis of an auxin/indole-3-acetic acid transcription factor, and two impart phenotypes indicative of an altered auxin response, including impaired root development. Microarray analysis was used to demonstrate the mechanistic similarities of the two most potent molecules. This strategy promises to yield powerful tools for the discovery of unidentified components of the auxin-signaling networks and the study of auxin's participation in various stages of plant development.