Background and Objective:Oxidative stress plays an important role in the pathogensis of various diseases, including lung cancer, chronic obstructive pulmonary disease, and atherosclerosis. Total antioxidant capacity plays a significant role in the body's antioxidant defense, so its assessment is a matter of the utmost importance. Currently, assessment of this capacity is performed in various scientific fields by expensive imported kits. The aim of the present study was to design a sensitive fluorometric method for the assessment of total antioxidant capacity and improvement of sensitivity, accuracy, and speed of the measurement.Methods: The sensitivity and intra-and inter-assay accuracy, verification by recovery and parallelism tests, method comparison, and correlation and coherence evaluation were performed. To increase the accuracy and speed of reading, the assay was performed in a microplate and reading was done using a fluorometer plate.Result: In accuracy assessment, intra-and inter-assay coefficient of variation was calculated to be 4.1-5.7 and 4.4-7.5, respectively. In validity evaluation, the recovery percentage was calculated to be 90-109, the recovery percentage range was 90 to 109. Comparison of the results of this method on 50 serum samples with common colorimetric method, indicated a good correlation (0.93). The sensitivity of the studied method was 0.01 mM/l.Conclusion: Microplate-reader fluorometry, in addition to increasing the speed of measurement, has enough efficacy, accuracy, and sensitivity to assess total antioxidant capacity and could be an appropriate alternative for current colorimetric methods.