Three transgenic soybean lines expressing the Cry1Ia5 gene were developed using the Agrobacterium transformation system. The integration of the Cry1Ia5 gene in the genome of the transgenic plants was approved using specific primers for PCR and real time PCR analysis, respectively. The insecticidal activity of three transgenic lines (L1, L2 and L3) against 2nd larval instars Spodoptera littoralis was tested. The data indicate that L2 exhibited the highest mortality percentage 9 days post feeding (60%) followed by L3 (40%) then L1 (20%) while the control showed 0% mortality. The larvae fed transgenic material appeared smaller in size than compared to the control larvae. The reduction in insect size and weight was due to the accumulation of higher phenoloxidase activity in insect tissues. The higher mortality observed in L2 was due to a significant decrease in the acetylcholine esterase activity that leads to accumulation of acetylcholin at higher levels which causes paralysis and death. The developed transgenic line 2 could be used to construct an insect resistant soybean cultivar.