Divakar Gupta,1,* Joanne C Wen,2,* Janet L Huebner,3 Sandra Stinnett,1 Virginia B Kraus,3,4 Henry C Tseng,1 Molly Walsh1 1Department of Ophthalmology, Duke University Medical Center, Durham, NC, 2Department of Ophthalmology, University of Washington, Seattle, WA, 3Duke Molecular Physiology Institute, 4Division of Rheumatology, Department of Medicine, Duke University School of Medicine, Durham, NC, USA *These authors contributed equally to this work Purpose: To determine the utility of tear film cytokines as biomarkers for early primary open-angle glaucoma (POAG). Methods: Patients without POAG and eye drop-naïve patients with newly diagnosed POAG were recruited from an academic hospital-based glaucoma practice. Tear films of recruited patients were obtained and analyzed using a multiplex, high-sensitivity electrochemiluminescent enzyme-linked immunosorbent assay for proinflammatory cytokines (IFNγ, IL-10, IL-12p70, IL-13, IL-1β, IL-2, IL-4, IL-6, IL-8, and TNFα). Results: Mean concentrations of tear film cytokines were lower in the glaucoma group for 8 of 10 cytokines tested. IL-12p70 (3.94±2.19 pg/mL in control vs 2.31±1.156 pg/mL in POAG; P=0.035) was significantly lower in the tear film of patients with newly diagnosed POAG. Conclusion: Proinflammatory cytokines were lower in eye drop-naïve newly diagnosed glaucoma patients. Tear film cytokine profiles may be used as biomarkers of early POAG. Keywords: glaucoma, biomarkers, tear film, cytokines, glaucoma diagnosis, lower limit of detection