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Aim: To directly quantify peroxynitrite (ONOO - ) using a highly sensitive fluorescence resonance energy transfer probe RN-NA, investigate the association between ONOO - and primary open angle glaucoma (POAG), and clarify whether RN-NA could be used as a potential tool for POAG diagnosis., Methods: Plasma and aqueous humor (AH) samples were collected from POAG patients ( n =100, age: 59.70±6.87y) and age-related cataract (ARC) patients ( n =100, age: 61.15±4.60y) admitted to our hospital. Next, RN-NA was used to detect ONOO - in plasma and AH samples, and the relationship between ONOO - level and POAG was analyzed using binary logistic regression. Besides, Pearson correlation analysis was applied to characterize the correlation of the levels of ONOO - with the patients' age, intraocular pressure (IOP), and mean deviation of visual field testing. The ONOO - scavenger MnTMPyP was employed to treat the 3-morpholinosyndnomine (SIN-1)-induced ocular hypertension in mice ( n =7, 6-8wk). Finally, the IOP and ONOO - in both eyes were measured 30min after the last drug treatment., Results: ONOO - levels of AH and plasma were significantly higher in the POAG group than in the ARC group ( P <0.01). Additionally, ONOO - levels were closely correlated with POAG in a binary logistic regression analysis [odds ratio (OR)=1.008, 95% confidence interval (CI): 1.002-1.013, P <0.01 for AH; OR=1.004, 95%CI: 1.002-1.006, P <0.001 for plasma]. Pearson correlation analysis showed that ONOO - levels in AH or plasma were positively associated with visual field defects ( R =0.51, P <0.01 for AH; R =0.45, P <0.001 for plasma), and ONOO - levels in plasma and AH were correlated in the POAG group ( R =0.69, P <0.001). However, administering MnTMPyP to mouse eyes reversed the elevated IOP caused by SIN-1 ( P <0.05)., Conclusion: ONOO - levels in AH and plasma, detected by RN-NA, are significantly related to POAG and positively correlated with visual field defects in POAG patients. Hence, ONOO - is a potential biomarker of POAG, especially advanced POAG. Besides, anti-nitration compounds may be novel ocular hypotensive agents based on the animal study., Competing Interests: Conflicts of Interest: Qin B, None; Lei Y, None; Bao L, None; Zhang YJ, None; Liu KY, None; Chen YH, None., (International Journal of Ophthalmology Press.)