Low levels of synthetic auxins are used as root growth promoters for a wide variety of botanicals. When properly used, they can produce earlier and more prolific root growth. Indole-3-succinic acid (ISA) is a chiral compound that can be synthesized as a racemate. Its enantiomers were resolved by both chromatography and diastereomeric crystallization. The absolute configuration of each enantiomer was determined by X-ray crystallography and then tested for auxin activity. The relative effectiveness of the ISA enantiomers was determined and compared to the achiral synthetic auxin indole-3-butyric acid and to the natural auxin indole-3-acetic acid.