From studying diseases to evaluating toxic reactions induced by drugs and environmental compounds, cell-based assays typically focus on mitochondria and aim to monitor cellular health. Mitochondrial function is a key indicator of overall cellular health, and mitochondrial dysfunction is closely associated with various diseases, including Parkinson's disease, Alzheimer's disease, heart failure, ischemic diseases, and cancer. In addition, the pathogenesis of rare diseases may be driven by mutations in genes encoding mitochondrial proteins or mitochondrial DNA. Evaluating mitochondrial function is crucial throughout the entire drug development and testing process. The development of therapies to restore mitochondrial function and the development of chemotherapy drugs targeting cancer mitochondrial dysfunction are examples. In addition, the assessment of mitochondrial function is a crucial step in preclinical drug safety evaluation, as mitochondrial dysfunction may be triggered by drugs and have adverse effects on cellular and tissue health. Measuring mitochondrial membrane potential (MMP) to monitor changes in mitochondrial metabolic activity is a sensitive and effective method for evaluating mitochondrial function. Mitochondrial membrane potential is an important tool for detecting mitochondrial depolarization during apoptosis, detecting multidrug-resistant cells, and evaluating mitochondrial function in various toxicity screenings. The evaluation of 1-2 MMP is usually carried out using lipophilic cationic fluorescent dyes. According to the state of mitochondrial membrane potential, the fluorescence of MITO-ID membrane potential dye will change. Specifically, it exists as a monomer in the cytoplasm and emits green fluorescence (FITC, 530 nM); In active mitochondria, it aggregates in the mitochondrial matrix and emits orange fluorescence (TRITC, 570 nM). When MMP is disrupted, MITO-ID dye exits depolarized mitochondria and exists in monomeric form, and orange fluorescence can be observed to decrease to disappear.