Chemical biology is a scientific discipline between chemistry and biology. It provides various way to explore biological phenomena in the cellular level and involves the application of chemical techniques, analysis, and often small molecules produced through synthetic chemistry, to the study and manipulation of biological systems. In this study, we develop new theragnostic tools targeting mitochondria in Glioma cells. Because mitochondria are essential organelles for regulating energy homeostasis and intrinsic apoptosis, the perturbation of mitochondrial functions has been considered as an anti cancer treatment. Therefore, a new near-infrared (NIR) fluorescent probe, SiR-Mito11 targeting mitochondria was developed as a theragnostic agent and then was applicable Chemical biology is a scientific discipline between chemistry and biology. It provides various way to explore biological phenomena in the cellular level and involves the application of chemical techniques, analysis, and often small molecules produced through synthetic chemistry, to the study and manipulation of biological systems. In this study, we develop new theragnostic tools targeting mitochondria in Glioma cells. Because mitochondria are essential organelles for regulating energy homeostasis and intrinsic apoptosis, the perturbation of mitochondrial functions has been considered as an anti cancer treatment. Therefore, a new near-infrared (NIR) fluorescent probe, SiR-Mito11 targeting mitochondria was developed as a theragnostic agent and then was applicable for brain tumor models. SiR-Mito11 exhibited a potential anti-cancer activity against glioma cells, but was tolerant in normal neuronal cells. We further confirmed that the selective accumulation of SiR-Mito11 in glioma cells disrupted mitochondria membrane potential, followed by apoptotic cell death. In conclusion, through this approach, it is possible to treat various brain tumor. This study will contribute to understanding of the complex biological phenomena for targeted therapies.