Redox homeostasis is crucial for the maintenance of organism fitness and survival. Redox imbalance is a marker of various pathologies including cancer and neurodegenerative diseases. At high levels, reactive electrophilic/oxygen/nitrogen species cause damage to cellular components such as proteins and DNA. More recently, it has come to light that at physiological concentrations the reactive species act as signaling molecules crucial for cellular communication. Given the chemical simplicity of these reactive messengers, it has been a challenge to understand how these reactive small molecules specifically perturb particular proteins—a prerequisite of redox signaling. Traditionally, redox signaling has been studied by swamping a model system (cells/whole organisms) with reactive signals leading to the generation of mixed responses from multiple simultaneous events. Such approaches mimic oxidative stress and are less amenable to the study of redox signaling. Here, I report the development and characterization of the targetable reactive electrophiles and oxidants (T-REX), a unique chemistry-based platform that (1) enables selective modification of redox-sensitive proteins with spatiotemporal precision in complex biological systems, (2) interrogates the consequences of this target-specific redox modification, and (3) allows unbiased screening for novel first-responding sensors capable of sensing reactive redox signals under signal-limited conditions. As proof-of-concept, I show that T-REX can selectively modify Keap1, an established electrophile-sensitive protein and an important regulator of the therapeutically-relevant Nrf2/antioxidant response (Nrf2/AR) signaling axis, with the model electrophile 4-hydroxynonenal (HNE). Additionally, my work for the first time shows that low stoichiometry HNE modification of Keap1 is sufficient to trigger AR in biological systems. This work also expands the applicability of T-REX to study redox signaling in zebrafish (Z-REX) and E. coli. I report here that selective modification of Keap1 in zebrafish suppresses innate and adaptive immune response. Finally, my collaborative work also shows that T-REX can be used to screen for novel first-responding redox sensors. I show that Akt3, an isoform of the Akt oncogenic kinase, senses electrophilic signals using a unique cysteine residue in the flexible linker region of the enzyme. HNE modification of Akt3 downregulates its kinase activity with functional signaling consequences in cells and zebrafish.