Bioluminescence is a remarkable phenomenon in which living organisms emit light as a result of a specific biochemical reaction. In recent years, bioluminescent proteins have garnered considerable attention owing to their potential applications in a variety of fields, including biosensors, biothreat assessment, and notably, as a sought-after reporter gene to observe gene expression, promoter activity, post-translational regulation assays. as well as in the realm of protein engineering. Organisms with bioluminescence properties produce light through the oxidation of a substrate known as luciferin, a reaction that is catalyzed by a specific class of enzymes referred to as luciferases. Among the array of bioluminescent proteins, Renilla luciferase (RLuc) is particularly appropriate for its applications as bioluminescent tags. Unlike other bioluminescent proteins that depend on ATP, these enzymes function without ATP reliance. Instead, they require only the substrate and molecular oxygen, setting them apart in the field of bioluminescence. This 36 kDa enzyme, derived from the sea pansy, Renilla reniformis catalyzes the oxidation of the substrate coelenterazine, an aromatic compound specifically characterized as 2-(phydroxybenzyl)-6-(p-hydroxyphenyl)-8-benzylimidazo(1,2-a)pyrazine-3-(7H)-one. During the catalytic process, the substrate undergoes oxidative decarboxylation reaction, which results in the opening of the imidazole ring. This step initiates the release of carbon dioxide and the production of coelenteramide in the first singlet excited state (S1). The return of coelenteramide to the ground state (S0) triggers the release of blue light, a phenomenon known as bioluminescence. (see Scheme 1).