1 This is a post-peer-review, pre-copyedit version of an article published in Methods in Molecular Biology. The final authenticated version is available online at: https://doi.org/10.1007/978-1-62703-592-7_1 Fully-automated compound screening in Arabidopsis thaliana seedlings Dominique Audenaert 1,2 , Long Nguyen 3 , Bert De Rybel 4 , Tom Beeckman 1,2 1 Department of Plant Systems Biology, VIB, Technologiepark 927, B-9052 Gent, Belgium 2 Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 927, B-9052 Gent, Belgium 3 VIB Compound Screening Facility (VIB-CSF), Technologiepark 927, B-9052 Gent, Belgium 4 Current address: Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands Abstract High-throughput small molecule screenings in model plants are of great value to identify compounds that interfere with plant developmental processes. In academic research, the plant Arabidopsis thaliana is the most commonly used model organism for this purpose. However, compared to plant cellular systems, Arabidopsis thaliana plants are less amenable to develop high-throughput screening assays. In this chapter, we describe a screening procedure that is compatible with liquid handling systems and increases the throughput of compound screenings in Arabidopsis thaliana seedlings. 1. Introduction In chemical biology, low molecular mass molecules are applied as conditional tools to reveal the underlying mechanisms that control growth and development. Chemical biology approaches have been well established to study human biology and disease mechanisms (1, 2). Also in plant sciences, several studies have shown that chemical biology represents a powerful tool to study plant developmental processes (3, 4). The identification of small molecules that interfere with a biological process of interest requires screening of a large number of compounds in an assay that is compatible with high- throughput screening. During the past two decades, affordable collections of small