Intercellular host-associated bacteria shape the chemistry of the living eukaryotic environment. Pathogenic microorganisms like barley-and wheat-associated Xanthomonas translucens, swiftly overtake the inner leaf tissue becoming the dominant community member during disease development. To define the metabolic signals associated with inner leaf colonization, we used untargeted metabolomics to characterize Xanthomonas translucens pathovars translucens (Xtt) and undulosa (Xtu)primary metabolism signatures associated with mesophyll growth. This protocol indicates how to perform the infections, extract apoplastic fluids, perform GCMS, and conduct the subsequent data analysis.