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Inter- and intraday precisions in all samples were both less then 12.9% and the accuracy was within 92.1%-108.4%. The highest concentration of tropicamide was found in aqueous humor (Cmax 29430 ng/g), while was in cornea for phenylephrine (Cmax 3465 ng/g). All the ocular tissues concentrations were much higher than those of blood. Conclusion This UPLC-MS/MS method allowed us to determine the PK and distribution of tropicamide and phenylephrine in rabbit ocular tissue, which may be helpful in the future development and application of myd