Burn injury can be a devastating traumatic injury, with long-term personal and social implications for the patient. The many complex local and disseminating pathological processes underlying burn injury's clinical challenges are orchestrated from the site of injury and develop over time, yet few studies of the molecular basis of these mechanisms specifically explore the local signaling environment. Those that do are typically destructive in nature and preclude the collection of longitudinal temporal data. Burn injury therefore exemplifies a superficial temporally dynamic pathology for which experimental sampling typically prioritizes either specificity to the local burn site or continuous collection from circulation. Here, we present an exploratory approach to the targeted elucidation of complex, local, acutely temporally dynamic interstitia through its application to burn injury. Subcutaneous microdialysis is coupled with ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) analysis, permitting the application of high-throughput metabolomic profiling to samples collected both continuously and specifically from the burn site. We demonstrate this workflow's high yield of burn-altered metabolites including the complete structural elucidation of niacinamide and uric acid, two compounds potentially involved in the pathology of burn injury. Further understanding the metabolic changes induced by burn injury will help to guide therapeutic intervention in the future. This approach is equally applicable to the analysis of other tissues and pathological conditions, so it may further improve our understanding of the metabolic changes underlying a wide variety of pathological processes.