AbstractWe present a simple one‐pot extraction protocol, which rapidly isolates hydrophilic metabolites, lipids, and proteins from the same pulverized plant sample. Also detailed is a global plant proteomics sample preparation method utilizing iTRAQ multiplexing reagents that enables deep proteome coverage due to the use of HPLC fractionation of the peptides prior to mass spectrometric analysis. We have successfully used this protocol on several different plant tissues (e.g., roots, stems, leaves) from different plants (e.g., sorghum, poplar, Arabidopsis, soybean), and have been able to successfully detect and quantify thousands of proteins. Multiplexing strategies such as iTRAQ and the bioinformatics strategy outlined here, ultimately provide insight into which proteins are significantly changed in abundance between two or more groups (e.g., control, perturbation). Our bioinformatics strategy yields z‐score values, which normalize the expression data into a format that can easily be cross‐compared with other expression data (i.e., metabolomics, transcriptomics) obtained from different analytical methods and instrumentation. © 2017 by John Wiley & Sons, Inc.