Mitochondria are vital organelles to eukaryotic cells. Damage and dysfunction of mitochondria cause cell apoptosis and consequent cell death. Hence, mitochondrial specific targeting gains lots of interest in cancer therapeutics at present. Delivery of cancer specific drug into mitochondria has already been established in recent years by various approaches such as conjugation of drug molecule with MPPs and other mitochondria targeting ligands.1.2 But mitochondrial damage due to self-assembly of amphiphilic molecules inside mitochondria still remains challenging. It is already well proved that peptides have intrinsic ability to form various selfassembled structure such as fiber, ribbon, and sphere. Study of selfassembling behavior of peptide inside the live cell organelle is least established till now.3,4 Here, we introduce mitochondria targeting short peptide containing sequence as PyBuFFK(TPP) [PyBu = Pyrenebutyric acid, TPP = Triphenyl phosphonium, F = phenyl alanine, K = lysine]. The TPP moiety can provide mitochondrial specific targeting and pyrene butyric acid provides both self-assembling property and can act as a fluorescent probe. The peptide exhibited well-defined morphology in water, improved cytotoxicity and high mitochondrial localization. The improved mitochondrial localization of peptide is attributed to the TPP moiety and high hydrophobicity. Under the mitochondrial environment the peptide can undergo aggregation due to increased concentration above the CAC (40 uM), so that peptide undergo self-assembly and induce mitochondrial stress and thereby generate ROS and cause cell death. The effect of self-assembly on cellular toxicity has been studied by synthesizing a series of peptide with varying amino acid sequence.