Purpose: Dry eye disease (DED) is a chronic multifactorial disorder affecting millions of people, yet the pathogenesis mechanisms still remain unclear. Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) is a novel in situ visualization approach combined high-throughput mass spectrometry and molecular imaging. We aimed to explore the in situ ocular metabolic changes via MALDI-MSI to accelerate the recognition of DED pathogenesis.Methods: Experimental dry eye was established in Wistar rats by subcutaneous injection of scopolamine. The induction of DED was assessed by tear film breakup time, sodium fluorescein, histopathological staining and cell apoptosis. MALDI-MSI was applied to explore in situ ocular metabolomic in DED rats, and histopathological staining from same sections were used for side-by-side comparison with MALDI to annotate different tissue structures in the eye.Results: Considering the complexity of ocular tissue, we visualized the metabolites in specific ocular regions (central cornea, peripheral cornea, fornix conjunctiva, eyelid conjunctiva and aqueous humor), and identified metabolites related to DED, with information of relative abundance and spatial signatures. In addition, integrative pathway analysis illustrated that, several metabolic pathways such as glycerophospholipid, sphingolipid phenylalanine, and metabolism of glycine, serine and threonine were significantly altered in certain regions in the dry eye tissue. Moreover, we discussed how the metabolic pathways with spatiotemporal signatures might be involved in the DED process.Conclusions: Our data exploit the advantages of in situ analysis of MALDI-MSI to accurately analyze the region specific metabolic behaviors in DED, and provide new clues to uncover DED pathogenesis.