Glutathione (GSH) plays an important role in the body's biochemical defense system, and the detection of GSH in a physiological system is an important tool for understanding redox homeostasis. Protection-deprotection strategies have proven to be the most reliable, among existing detection methods. However, the understanding of how various electronic and steric effects influence a probe's ability to recognize a substrate is still lacking. In this study, we have analyzed various substituent effects on a GSH probe template via theoretical calculations and constructed the performance regulation and control strategy for this kind of probe. We then developed a series of guided probes using eighteen different acrylic ester derivatives to mask the fluorescence of fluorescein. The optical performance differences between the guided probes strongly supported the applicability of our proposed guiding strategy. Moreover, the positively guided probes are excellent for imaging GSH distribution in living cells and mice.