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Lipid metabolism and signaling play pivotal functions in biology and disease development. Despite this, currently available optical techniques are limited in their ability to directly visualize the lipidome in tissues. In this study, opto‐lipidomics, a new approach to optical molecular tissue imaging is introduced. The capability of vibrational Raman spectroscopy is expanded to identify individual lipids in complex tissue matrices through correlation with desorption electrospray ionization (DESI) – mass spectrometry (MS) imaging in an integrated instrument. A computational pipeline of inter‐modality analysis is established to infer lipidomic information from optical vibrational spectra. Opto‐lipidomic imaging of transient cerebral ischemia‐reperfusion injury in a murine model of ischemic stroke demonstrates the visualization and identification of lipids in disease with high molecular specificity using Raman scattered light. Furthermore, opto‐lipidomics in a handheld fiber‐optic Raman probe is deployed and demonstrates real‐time classification of bulk brain tissues based on specific lipid abundances. Opto‐lipidomics opens a host of new opportunities to study lipid biomarkers for diagnostics, prognostics, and novel therapeutic targets. [ABSTRACT FROM AUTHOR]