Singlet oxygen (1O2) is a type of reactive oxygen species involved in numerous physiological activities. We previously reported that 1O2-specific oxidation products are increased in patients with prediabetes, suggesting that measurement of 1O2 may be an important indicator of physiological and pathological conditions. The turnover in the generation and quenching of 1O2 is extremely rapid during biological activities owing to it high reactivity and short lifetime in solution. However, the dynamic changes in 1O2 generation in living cells have not been fully explored. In this study, we investigated whether the kinetics of 1O2 generation can be quantified using a far-red fluorescent probe for mitochondrial 1O2, Si-DMA, following addition of the 1O2 generator, endoperoxide, to mammalian cells. The kinetics of Si-DMA fluorescence intensity dose-dependently increased following treatment of mammalian living cells with endoperoxide. Alternatively, treatment with 1O2 quenchers decreased the fluorescence intensities following endoperoxide treatment. Our results indicate that the kinetics of intracellular 1O2 can be readily obtained using Si-DMA and time-lapse imaging, which provides new insights into the mechanism of 1O2 generation in mammalian cells and the exploration of 1O2 generators and quenchers.