We report a ratiometric two-photon probe (SSH-Mito) for mitochondrial thiols. This probe shows a marked blue-to-yellow emission color change in response to RSH, a significant two-photon cross section, good mitochondrial thiol selectivity, low cytotoxicity, and insensitivity to pH over the biologically relevant pH range, allowing the direct visualization of RSH levels in live cells as well as in living tissues at 90-190 mu m depth without interference from other biologically relevant species through the use of two-photon microscopy.