Abstract: Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) is an important polyphagous pest in the Mediterranean area. However, insecticidal proteins produced by genetically modified (GM) plants have sublethal effects on their caterpillars. We used stacked GM maize expressing lepidopteran active Cry1F, Cry1A.105, and Cry2Ab2 proteins from Bacillus thuringiensis (Bt) to explore the synergic effect on S. littoralis. We designed 48-hour long bitrophic study to assess the effect of three lepidopteran active Cry proteins on mortality of S. littoralis caterpillars. The second aim of our study was to investigate whether the different Cry proteins dilute differently when they are transferred from the plant to S. littoralis. More than 98% mortality was observed for first, second, and third S. littoralis instars. All Cry proteins were diluted when transferred from maize to S. littoralis. Cry1A.105 was the least reduced Cry protein detected in S. littoralis while Cry2Ab2 was the most diluted Cry protein despite the fact that it was the most expressed Cry protein in leaves. We conclude that stacking of three lepidopteran active Cry proteins cause high mortality to S. littoralis in contrast to only sublethal effects reported in the literature for GM maize expressing single Cry proteins. Spodoptera species have been often used in trophic experiments as a source of Cry proteins to natural enemies. However, high mortality of S. littoralis caterpillars fed stacked Bt maize prevents the use of this species as a source of Cry proteins in feeding bioassays with natural enemies.