Mitochondria are vital organelles to eukaryotic cells. Since damage and dysfunction of mitochondria cause cell apoptosis, mitochondrial specific targeting gains lots of interest in cancer therapeutics. Delivery of cancer specific drug into mitochondria has already been established in recent years by various approaches such as conjugation of drug molecule with mitochondria targeting ligands. However, 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 self-assembled structure such as fiber, ribbon, sphere and vesicle. Study of selfassembling behavior of peptide inside the live cell organelle is least established till now. Here, we introduce mitochondria targeting short peptide containing sequence as Py-FFK(TPP) [Py = Pyrenebutyric acid, TPP = Triphenyl phosphonium, F = phenyl alanine, K = lysine]. The TPP moiety can provide mitochondrial specific targeting to mitochondria and pyrene butyric acid provides both a self-assembling building block and a fluorescent probe. The peptide exhibited well-defined morphology in water, high mitochondrial localization and cancer-cell cytotoxicity. 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 critial aggregation cocnetraion (40 uM). The self-assembly of the peptide induces mitochondrial stress and thereby generates ROS and causes cell death.