<p>This study evaluated the physicochemical properties of the commercial Rhopressa formulation and investigated the dose–response effects of Rhopressa-equivalent dilutions on cell viability, wound healing, and inflammatory signaling in human corneal epithelial (HCE) cells under static in vitro conditions. Rhopressa exhibited an acidic pH of 5.5 and an osmolarity of 275 mOsm/kg, remaining within a near-physiological tonicity range. Exposure to higher Rhopressa-equivalent dilutions (≥ 1% v/v) induced marked cytotoxicity and ultrastructural degeneration characterized by chromatin condensation and cytoplasmic vacuolization. In contrast, an optimal sublethal response zone was identified at the 0.5% Rhopressa-equivalent dilution, which significantly accelerated corneal epithelial wound closure. In a lipopolysaccharide (LPS)-induced injury model, the 0.5% Rhopressa-equivalent dilution exerted protective anti-inflammatory effects by suppressing intracellular reactive oxygen species (ROS) generation and attenuating NF-κB nuclear translocation. These findings demonstrate biphasic epithelial stress–response patterns to Rhopressa-equivalent exposure and provide mechanistic insight for future hypothesis-driven studies on ocular surface safety and formulation-based dosing optimization.</p>