The present study was undertaken to understand the protease mediated resistance mechanism of Cry1C and Vip3A in S. litura.  Proteases play an important role for convertion of protoxins to active toxin. Hence laboratory selection of Spodoptera litura (Fab.) with Cry1C and Vip3A was done for eight generations to develop resistance, which developed 30.32 and 285.47 fold resistance respectively.  Fourth instar larvae from nine generation of S. litura were dissected in an ice-cold 20 mM Tris-HCl buffer (pH7). Major midgut proteases viz, azocaseinolytic, tryptic and chymotryptic were quantified by using azocasein, trypsin and chymotrypsin as substrate. Results revealed that lowest azocaseinolytic activity 0.896 U/gut was observed in Cry1C resistant strain of S. litura which was 2.57 fold less than susceptible. Similarly, Vip3A resistant strain recorded 1.08 U/gut azocaseinolytic activities, which was 2.13 fold less than susceptible strain of S. litura. There was 2.26 and 3.35 fold decrease in activity respectively in Cry1C resistant and Vip3A resistant S. litura over susceptible strains. Chymotryptic activity in midgut of susceptible stain of S. litura was 1.13 U/gut, whereas in Cry1C resistant strain it was 0.512 U/gut which was 2.20 fold less over susceptible strain. Similarly, in Vip3A resistant strain, lower chymotryptic activity was recorded as compared to susceptible, which was 1.66 fold less than susceptible strain. Maximum five protease isoforms were identified in the electrophoretic profile of susceptible S. litura. Remarkable variation was observed between susceptible and resistant S. litura for protease isoform. The Cry1C resistant S. litura homogenate showed three bands viz., Pro3, Pro4 and Pro5 while only two bands viz., Pro3 and Pro5  in Vip3A resistant homogenate of S. litura whereas five bands were observed in susceptible strain of S. litura.