Purpose: To evaluate the concentrations of brimonidine and brinzolamide in the vitreous and aqueous humor after instillation of a 0.1% brimonidine tartrate and 1% brinzolamide fixed-combination ophthalmic suspension. Methods: The present investigation involved patients with macular holes or idiopathic epiretinal membranes who were planning to undergo vitrectomy. One week prior to surgery, the patients received twice-daily topical treatment with 0.1% brimonidine tartrate and 1% brinzolamide fixed-combination ophthalmic suspension. Before vitrectomy, vitreous and aqueous humor samples were collected, and the mean concentrations of brimonidine and brinzolamide were determined through liquid chromatography-tandem spectrometry. Results: Ten eyes (nine phakic and one pseudophakic eyes; 10 patients) were examined. The concentration of brimonidine in vitreous and aqueous humor samples was 5.02 ± 2.24 and 559 ± 670 nM, respectively. The concentration of brimonidine in the vitreous humor, which is needed to activate α2 receptors, was >2 nM in all patients. The concentration of brinzolamide was 8.96 ± 4.65 and 1100 ± 813 nM, respectively. However, there was no significant correlation between the concentrations of brimonidine in the vitreous and aqueous humor samples. Conclusions: Sufficient concentrations of brimonidine were detected in all vitreous samples. The dissociated correlation of the drug concentrations between aqueous and vitreous humors implies the possibility of another pathway to vitreous humor, different from the pathway to aqueous humor.