Spodoptera litura is classified as a plant-disturbing organism. Efforts to control S. litura by using chemical insecticides have a detrimental impact on the environment and the potential to harm non-target organisms. Bioinsecticides provide a safe alternative for reducing the agricultural pest problem. The purpose of this study was to investigate the specific amino acid from Mirabilis jalapa extract using high performance-liquid chromatography (HP-LC) analysis and to identify their potency as a biopesticide. Mirabilis jalapa extract in combination with Bacillus thuringiensis influences the weakening of the S. litura immune system to explain the cause of S. litura death. The results showed that the M. jalapa extract had seven sequences of the highest amino acid compounds from M. jalapa, namely: Glu, Asp, Lys, Val, Leu, Arg, and Ala. Alanine compound has the potential as a biopesticide that breaks down the muscle and nervous system and blocks its receptors. The combination of B. thuringiensis in LC₅₀ concentration also caused the death of S. litura. Finally, the combination of 0.2% concentration of M. jalapa and B. thuringiensis at a sublethal concentration (1.07%) applied in 12-hour intervals within 24 hours showed the optimum mortality of S. litura (p<0.05): the death of larvae was characterized by damage to the midgut organs in the digestive tract observed by the histological microanatomy.