AbstractTo fully exploit the potential of plant microbiome alterations to improve plant health, reliable methods must be used to prepare and characterize microbiome samples. The power of culture‐independent studies is that they allow the characterization of novel microbial community members, but only microbial members consistently represented between different research groups are likely to become broadly applicable treatments. The identification of plant microbiome members can be affected by several experimental stages, including design, sample preparation, nucleic acid extraction, sequencing, and analysis. The protocols described here therefore aim to highlight crucial steps that experimenters should consider before beginning a plant microbiome study. © 2017 by John Wiley & Sons, Inc.