Phenoloxidase activity was detected in the serum of Spodoptera litura, which is present as an inactive proenzyme, proPhenoloxidase. Substrate screening was done using different phenolic substrates, in which the PO was high in L-DOPA at 458nm. S. litura was treated with fungal pathogens Beauveria bassiana and Metarhizium anisopliae, Compared to untreated S. litura, the proPhenoloxidase activity was high in fungal infected S. litura. The fungal pathogen also induced pupal and adult deformities. In both untreated and treated S. litura, on addition of activators effectively triggers the proPO activation. The addition of protease inhibitor, chelators and eicosonaids constantly inhibited the activation of proPO, in which PTU, DTT and DEX shows effective inhibition of proPO to PO, EDTA shows little effect even in higher concentration. Malformed pupae and emergence of adults with crippled wings strongly suggest the efficacy of the selected fungi as biocontrol agent.