Shotgun analysis provides a quantitative snapshot of the lipidome composition of cells, tissues, or model organisms; however, it does not elucidate the spatial distribution of lipids. Here we demonstrate that shotgun analysis could quantify low-picomole amounts of lipids isolated by laser capture microdissection (LCM) of hundred micrometer-sized histological zones visualized at the cryosections of tissues. We identified metabolically distinct periportal (pp) and pericentral (pc) zones by immunostaining of 20 μm thick cryosections of a healthy mouse liver. LCM was used to ablate, catapult, and collect the tissue material from 10 to 20 individual zones covering a total area of 0.3-0.5 mm2 and containing ca. 500 cells. Top-down shotgun profiling relying upon computational stitching of 61 targeted selective ion monitoring ( t-SIM) spectra quantified more than 200 lipid species from 17 lipid classes including glycero- and glycerophospholipids, sphingolipids, cholesterol esters, and cholesterol. Shotgun LCM revealed the overall commonality of the full lipidome composition of pp and pc zones along with significant ( p < 0.001) difference in the relative abundance of 13 lipid species. Follow-up proteomics analyses of pellets recovered from an aqueous phase saved after the lipid extraction identified 13 known and 7 new protein markers exclusively present in pp or in pc zones and independently validated the specificity of their visualization, isolation, and histological assignment.