The upland cotton (Gossypium hirsutum) belongs to the family Malvaceae and cultivated since ancient times in warmer parts of the world. Pakistan is the 4th largest cotton producing country and prominent cotton yarn exporter of the world. Cotton is the major source of natural fiber and significantly contributes to the economy of Pakistan. There are many factors (biotic and abiotic) affecting the cotton yield in Pakistan. The cotton leaf curl disease (CLCuD) is one of the biotic factors and endemic in the most cotton growing areas of the country. The antioxidant enzymes and antioxidant biochemical metabolites play important role during stress. The present study was planned to compare the antioxidant enzymes and metabolites from healthy and CLCuD infected cotton leaves. Antioxidant enzymes activities including catalase, peroxidase and peroxidase were measured through different antioxidant assays and different antioxidant metabolites were also determined. During stress condition antioxidant metabolites serve as signal for the activation of antioxidant enzymes. Present study revealed that antioxidant enzymes were significantly higher in the infected cotton leaves as compared to the healthy cotton leaves. While the amount of antioxidant metabolites like total phenolic contents (TPC) and total flavonoid contents (TFC) were higher in healthy cotton leaves as compared to the infected leaves.