With the recent advances of sensitive detection technology such as positron-emission tomography (PET) and fluorescent spectroscopy, there has been the demand for diverse imaging probes including fluorescent probes. Xanthene fluorophores including fluorescein, rhodamine and their hybrid structure, rhodol, are among the most commonly used fluorescent probes, and many activity-based fluorescent probes have been discovered with their asymmetric derivatives in many areas including biology and medicines. Of these fluorescent probes with xanthene structure, rhodol derivatives have been of great interest as activity-based fluorescent probes that can detect various phenonmena in live cells as well as metal ions because rhodol probes are fluorescent with a high quantum yield in aqueous solution within broad ranges of pH and are also known to be photo-stable as compared to other fluorescent probes such as fluorescein. However, as with fluorescein and rhodamine probes, rhodol probes also have a synthetic disadvantage that it’s difficult to modify and introduce diverse substituents at the terminal of xanthene ring due to low chemical stability to acids and bases. A few reduced fluorescein and rhodol fluorophores have been developed by several groups but the reduced rhodamine fluorescent probe has not been discovered. Herein, we describe a new reduced rhodamine fluorescent probes with chemical stability and its application to a new activity-based fluorescent probe to detect hypoxia.