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Purpose The purpose of this study was to detect the mechanical anisotropy of the cornea using Brillouin microscopy along different perturbation directions. Methods Brillouin frequency shift of both whole globes (n = 10) and cornea punches (n = 10) were measured at different angles to the incident laser, thereby probing corneal longitudinal modulus of elasticity along different directions. Frequency shift of virgin (n = 26) versus cross-linked corneas (n = 15) over a large range of hydration conditions were compared in order to differentiate the contributions to Brillouin shift due to hydration from those due to stromal tissue. Results We detected mechanical anisotropy of corneas, with an average frequency shift increase of 53 MHz and 96 MHz when the instrument probed from 0° to 15° and 30° along the direction of the stromal fibers. Brillouin microscopy did not lose sensitivity to mechanical anisotropy up to 96% water content. We experimentally measured and theoretically modeled how mechanical changes independent of hydration affect frequency shift as a result of corneal cross-linking by isolating an approximately 100 MHz increase in frequency shift following a cross-linking procedure purely due to changes of stromal tissue mechanics. Conclusions Brillouin microscopy is sensitive to mechanical anisotropy of the stroma even in highly hydrated corneas. The agreement between model and experimental data suggested a quantitative relationship between Brillouin frequency shift, hydration state of the cornea, and stromal tissue stiffness. Translational relevance The protocol and model validated throughout this study offer a path for comprehensive measurements of corneal mechanics within the clinic; allowing for improved evaluation of the long-term mechanical efficacy of cross-linking procedures.