AbstractAmong targeted proteomic techniques, AQUA‐MRM is considered as one of the most reliable for accurate protein quantitation. This method displays high sensitivity, specificity, and reproducibility compared to many common biochemical techniques by coupling the use of unique, heavy‐labeled peptide standards and triple‐quadrupole mass spectrometry. However, there are several important steps that are required for successful development and validation of a robust AQUA‐MRM assay. The following protocol outlines and details the key steps necessary for plant sample preparation as well as AQUA‐MRM development and validation, specifically for absolute quantitation of plant proteins in vivo. © 2018 by John Wiley & Sons, Inc.