Non-Nutritive Sweeteners (NNS) are low-calorie synthetic sweeteners that have negative effects on metabolic, physiological, behavior and reproduction. A present study has been undertaken to understand the effects of NNS on accessory gland traits and reproductive fitness in Drosophila melanogaster (D. melanogaster). Dietary restrictions may lead to an imbalance in the production of Reactive Oxygen Species (ROS) and detoxification, which contribute to nutritional stress. A high level of oxidative stress affects the Accessory gland proteins (Acps) of D. melanogaster during reproduction. D. melanogaster has a vital role in nutrition research where diet-induced stress responses and reproductive and life-history traits can be examined under defined experimental procedures. In this work, aspartame and sucralose are used as NNS and nutritive sweetener sucrose to examine the effects of oxidative stress (ROS, SOD and CAT) and reproductive fitness of D. melanogaster. The results reveal that NNS has a positive correlation between oxidative stress and the reproduction of flies. Aspartame-treated flies showed higher ROS levels and lower SOD and CAT antioxidant enzyme activity. ACPS and reproduction traits were reduced in NNS-treated flies, suggesting that oxidative stress may be susceptible to the cost of reproduction.