A method comprising: a. providing to a cell: i. a luciferase enzyme protein sensor, wherein the protein sensor generates a detectable signal upon interaction with a substrate, and wherein the protein sensor is incapable of entering the cell; and ii. a stable pro-substrate that is capable of entering the cell wherein the stable pro-substrate comprises a pro-furimazine, pro-furimazine derivative, pro-coelenterazine, or pro-coelenterazine derivative or analog, wherein the interaction of the pro-substrate with an intracellular enzyme or molecule of interest converts the pro-substrate into an unstable substrate for the protein sensor, wherein the substrate is capable of exiting the cell, and also wherein the substrate is unstable due to inherent chemical instability and/or is rapidly enzymatically degraded by the protein sensor such that the substrate does not accumulate; and b. detecting the signal generated by the interaction of the substrate and the luciferase enzyme protein sensor by way of one or more of: real-time monitoring of the signal, continuous monitoring of the signal, and continuous monitoring of the signal over an extended period of time, wherein the magnitude of the signal depends on the rate of conversion of the pro-substrate such that the magnitude of the signal correlates with the intracellular amount of the enzyme or molecule of interest and changes in the rate of conversion of pro-substrate to substrate results in real-time changes in signal due to the unstable substrate. a. providing to a cell: i. a luciferase enzyme protein sensor, wherein the protein sensor generates a detectable signal upon interaction with a substrate, and wherein the protein sensor is incapable of entering the cell; and ii. a stable pro-substrate that is capable of entering the cell wherein the stable pro-substrate comprises a pro-furimazine, pro-furimazine derivative, pro-coelenterazine, or pro-coelenterazine derivative or analog, wherein the interaction of the pro-substrate with an intracellular enzyme or molecule of interest converts the pro-substrate into an unstable substrate for the protein sensor, wherein the substrate is capable of exiting the cell, and also wherein the substrate is unstable due to inherent chemical instability and/or is rapidly enzymatically degraded by the protein sensor such that the substrate does not accumulate; and i. a luciferase enzyme protein sensor, wherein the protein sensor generates a detectable signal upon interaction with a substrate, and wherein the protein sensor is incapable of entering the cell; and ii. a stable pro-substrate that is capable of entering the cell wherein the stable pro-substrate comprises a pro-furimazine, pro-furimazine derivative, pro-coelenterazine, or pro-coelenterazine derivative or analog, wherein the interaction of the pro-substrate with an intracellular enzyme or molecule of interest converts the pro-substrate into an unstable substrate for the protein sensor, wherein the substrate is capable of exiting the cell, and also wherein the substrate is unstable due to inherent chemical instability and/or is rapidly enzymatically degraded by the protein sensor such that the substrate does not accumulate; and b. detecting the signal generated by the interaction of the substrate and the luciferase enzyme protein sensor by way of one or more of: real-time monitoring of the signal, continuous monitoring of the signal, and continuous monitoring of the signal over an extended period of time, wherein the magnitude of the signal depends on the rate of conversion of the pro-substrate such that the magnitude of the signal correlates with the intracellular amount of the enzyme or molecule of interest and changes in the rate of conversion of pro-substrate to substrate results in real-time changes in signal due to the unstable substrate.