Purpose: To conduct a multi-tissue investigation on the penetration and distribution of topical atropine in myopia treatment, and determine if atropine is detectable in the untreated contralateral eye after uniocular instillation. Methods: Nine mature New Zealand white rabbits were evenly divided into three groups. Each group was killed at 5, 24 and 72 hr, respectively, following uniocular instillation of 0.05 ml of 1% atropine. Tissues were sampled after enucleation: conjunctiva, sclera, cornea, iris, ciliary body, lens, retina, aqueous, and vitreous humors. The assay for atropine was performed using liquid chromatography-mass spectrometry (LC-MS), and molecular tissue distribution was illustrated using matrix-assisted laser desorption ionization-imaging mass spectrometry (MALDI-IMS) via an independent experiment on murine eyes. Results: At 5 hr, the highest (mean AE SEM) concentration of atropine was detected in the conjunctiva (19.05 AE 5.57 ng/mg, p < 0.05) with a concentration gradient established anteriorly to posteriorly, as supported by MALDI-IMS. At 24 hr, preferential binding of atropine to posterior ocular tissues occurred, demonstrating a reversal of the initial concentration gradient. Atropine has good ocular bioavailability with concentrations of two magnitudes higher than its binding affinity in most tissues at 3 days. Crossing-over of atropine to the untreated eye occurred within 5 hr post-administration. Conclusion: Both transcorneal and transconjunctival-scleral routes are key in atropine absorption. Posterior ocular tissues could be important sites of action by atropine in myopic reduction. In uniocular atropine trials, cross-over effects on the placebo eye should be adjusted to enhance results reliability. Combining the use of LC-MS and MALDI-IMS can be a viable approach in the study of the ocular pharmacokinetics of atropine.