Peroxynitrite (ONOO-), generated from nitric oxide and superoxide, is implicated in cellular dysfunction and death.To detect ONOO- in live cells, we developed two hemicyanine-based ratiometric fluorophores, P-mito and P-boc.These ratiometric fluorophores were successfully used to monitor ONOO- fluctuations in cancer cells and endothelial cells.P-mito predominantly accumulated in the mitochondria of live cells and, upon selective reaction with ONOO-, increased the ratio of the fluorescence intensity from blue channel to that from red channel.Treatment with an ONOO- scavenger, N-acetyl cysteine, suppressed this ratio increase.Notably, in a model of endothelial dysfunction induced by treating pulmonary arterial endothelial cells with TGFβ2 and IL-1β, our system successful demonstrated increased ONOO- levels, effectively highlighting the utility of the hemicyanine-based fluorescence response to ONOO-.Therefore, our hemicyanine-based ONOO- detection system provides an effective platform for spatiotemporal monitoring and quant. anal. of ONOO- within live cells, enabling mol.-level research into various ONOO--related pathol. conditions.