To reveal the delicate function of mitochondria, spatiotemporally precise detection tools remain highly desirable. However, current probes with positively charged warheads for targeting mitochondria diffuse out of the mitochondria as the potential of the mitochondrial membrane changes, which directly influences the accuracy of the detection. Herein, we assembled carbonylpyridinium and chromene to afford the probe CM-Mit. Following the ultrafast response to thiol and the dissociation of carbonylpyridinium, the formation of -quinone methide from CM-Mit was proposed to label proteins, thus avoiding diffusion out of the mitochondria. Therefore, the accurate spatiotemporal detection of thiol in mitochondria was realized. With this excellent probe, ferroptosis inducers were proved to stimulate thiol starvation in mitochondria for the first time in cancer cells. Moreover, CM-Mit was used to screen a compound library developed in-house and the stemona alkaloid analog SA-11 was shown to induce ferroptosis in various cancer cell lines, including a drug-resistant one.