Genetically encoded fluorescent sensors have been widely applied to detect cell signaling molecules and events. We previously designed a fluorescent sensor motif suitable for detecting protease activity and opioids. In this manuscript, we demonstrated the motif's first use for reporting on protease activity in animal models, demonstrating a high signal-to-background ratio of 29. We further functionalized this sensor motif to detect the activity of the coronavirus main protease, Mpro, and demonstrated its utility in characterizing an Mpro inhibitor. The Mpro sensor will facilitate the study of coronaviral activity in cell cultures and potentially in animal models. Additionally, we developed an innovative method for engineering a protease-based time-gating mechanism using this versatile sensor motif, allowing the temporally controlled detection of opioids. This time-gating strategy for detecting opioids can be generalized to other similar sensors, enabling detection of G protein-coupled receptor ligands with improved temporal resolution.