There is growing interest in limiting the use of fungicides and implementing innovative, environmentally friendly strategies, such as the use of beneficial bacteria-triggered immunity, to protect grapevines from natural pathogens. Therefore, we need rapid and innovative ways to translate the knowledge of the molecular mechanisms underlying the activation of grapevine defenses against pathogens to induced resistance. Here, we have implemented an in vivo minimally invasive approach to study the interaction between plants and beneficial bacteria based on metabolic signatures. Paraburkholderia phytofirmans strain PsJN and PsJN–grapevine were used as bacterial and plant–bacterium interaction models, respectively. Using an innovative tool, SpiderMass, based on water-assisted laser desorption ionization with an IR microsampling probe, we simultaneously detect metabolic and lipidomic species. A metabolomic spectrum was thus generated, which was used to build a library and identify the most variable and discriminative peaks between the two conditions. We then showed that caftaric acid (m/z 311.04), caftaric acid dimer (m/z 623.09), derived caftaric acid (m/z 653.15), and quercetin-O-glucuronide tended to accumulate in grapevine leaves after root bacterization with PsJN. In addition, together with these phenolic messengers, we identified lipid biomarkers such as palmitic acid, linoleic acid, and α-linoleic acid as important messengers of enhanced defense mechanisms in Chardonnay plantlets. Taken together, SpiderMass is the next-generation methodology for studying plant–microorganism metabolic interactions with the prospect of in vivo real-time analysis in viticulture.