Dry eye disease (DED) is a multifactorial ocular surface disorder characterized by inflammation, oxidative stress, tear film instability, and secretory dysfunction. This study investigated the protective effects of Lute-gen^®, a lutein and zeaxanthin-based formulation, in both in vitro and in vivo DED models. Human corneal epithelial (HCE-T) cells were stimulated with TNF-α, while dry eye was induced in female Sprague–Dawley rats using subcutaneous scopolamine (SCP) administration. In HCE-T cells, Lute-gen^® showed no cytotoxicity, restored cell viability, reduced intracellular ROS, and was associated with increased expression of antioxidant-related markers (Nrf2, HO-1, SOD, CAT, and GPx), reduced expression of inflammatory mediators (TLR4/MyD88/NF-κB/NLRP3), and increased expression of AQP3 and AQP5. In SCP-induced rats, Lute-gen^® significantly improved tear secretion and reduced corneal fluorescein staining. Histopathological analyses revealed restoration of conjunctival goblet cells, mucin staining, corneal epithelial integrity, acinar area and cell density, and lacrimal gland architecture, with reduced inflammatory infiltration. Immunofluorescence further demonstrated reduced TLR4 and MMP9 immunoreactivity and decreased CD68⁺ inflammatory cell infiltration. Molecular analyses showed reduced expression of inflammatory cytokines and NF-κB/MAPK/MMP signaling-related inflammatory mediators, together with restoration of AQP1, AQP3, and AQP5 expression in corneal tissues. Collectively, these findings suggest that Lute-gen^® treatment was associated with improvements in dry eye-related pathological changes, including restoration of antioxidant-related markers, attenuation of inflammatory responses, restoration of aquaporin expression, and preservation of ocular surface and lacrimal gland integrity. These preclinical findings support further mechanistic investigations and the future clinical evaluation of Lute-gen^® as a potential nutritional intervention for dry eye disease.