Matrix-assisted laser desorption\/ionization\u2013imaging mass spectrometry (MALDI-IMS) evaluates drug distribution in biological samples. However, there have been few investigations of drug distribution in ocular tissues, including whole-eye segments. In the present study, we explored the spatial distribution of atropine in a whole-eye segment using MALDI-IMS. Atropine is a reversible muscarinic receptor used to treat various diseases. However, its distribution in ocular tissues remains unknown. A 1% atropine solution was administered to a rabbit and after 30 min, its eye was enucleated, sectioned, and analyzed by MALDI-IMS. Atropine accumulated primarily in the tear menisci but was found at substantially lower concentrations in the tissue surrounding the conjunctival sacs. Relative differences in atropine levels between the anterior and posterior regions provided insights into the post-instillation behavior of atropine. Atropine signal intensities differed among corneal layers and between the superior and inferior eyeball regions. Differences in signal intensity among tissues indicated that the drug migrated to the posterior regions via a periocular scleral route. This information is useful for atropine delivery and indicates that MALDI-IMS is effective for revealing drug distribution in whole-eye sections.