AbstractAuxin, a plant hormone, is able to stimulate growth and to control developmental processes. To get a deeper level of understanding of how auxin activity is regulated in planta, we have developed a sensitive and selective mass spectrometry‐based protocol for multiplex quantification of indole 3‐acetic acid (IAA) as well as its precursors and degradation products. As part of the protocol development, we have also established a derivatization protocol for the IAA precursors IPyA and IAAld, compounds that are highly labile during sample extraction and purification. This article describes a standard isotope dilution method for quantitative profiling of the IAA metabolome in small amounts of plant tissues. © 2016 by John Wiley & Sons, Inc.