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Spatial characterization of triglyceride metabolism is an area of significant interest which can be enabled by mass spectrometry imaging via recent advances in neutral lipid laser desorption analytical approaches. Here, we extend recent advancements in gold-assisted neutral lipid imaging and demonstrate the potential to map lipid flux in rodents. We address here critical issues surrounding the analytical configuration and interpretation of the data for a group of select triglycerides. Specifically, we examined how the signal intensity and spatial resolution would impact the apparent isotope ratio in a given analyte (which is an important consideration when performing MS based kinetics studies of this kind) with attention given to molecular ions and not fragments. We evaluated the analytics by contrasting lipid flux in well characterized mouse models, including fed vs fed states and different dietary perturbations. In total, the experimental paradigm described here should enable studies of hepatic lipogenesis; presumably, this logic can be enhanced via the inclusion of ion mobility and/or fragmentation. Although this study was carried out in robust models of liver lipogenesis, we expect that the model system could be expanded to a variety of tissues where zonated (or heterogeneous) lipid synthesis may occur, including solid tumor metabolism.