In an effort to attain more sustainable agricultural practices, there is a great interest in understanding metabolic processes within plant systems known to acquire nitrogen through biological nitrogen fixation. The symbiotic association between nitrogen-fixing soil bacteria (Rhizobiaceae) and plants of the family Leguminosae are one such system of interest. This symbiosis generates specialized organs, called root nodules, where rhizobia reduce N₂ into bioavailable products accessible to the host plant, and in exchange the plant provides a carbon source to the bacteria to ensure (among other things) sufficient energy for nitrogen fixation. Using both laser ablation electrospray ionization (LAESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) methods, we explored the array of metabolites involved, and their spatial distribution, that influence the rhizobia-legume association of Bradyrhizobium japonicum and soybean (Glycine max Williams 82). While these MS-based spatial metabolomics approaches provided insight into the heterogeneous distribution of analytes within soybean root nodules, orthogonal measurements were required for increased levels of confidence in the molecular identifications of the detected species. Here, we will describe how tandem MS, pre-mass analysis ion mobility separations, and high mass resolution and mass accuracy measurements of the isotopic envelope were utilized to provide confidence in the identity and localization of metabolites within soybean root nodules. We further applied this information to elucidate active metabolic pathways within different compartments of the nodules.