Transient luciferase reporter assays are a widely used and powerful tool for investigating promoter activity and gene regulation in plant systems. These assays make use of the enzymatic conversion of a luciferin substrate into bioluminescence to provide a highly sensitive and reproducible measure of transcriptional activity. The dual-luciferase system, incorporating Firefly luciferase (LUC) as the primary reporter and Renilla luciferase (REN) as an internal control, enhances experimental accuracy by normalizing for variations in infiltration efficiency, tissue viability, and environmental conditions. Agroinfiltration-mediated transient expression in Nicotiana benthamiana leaves enables rapid and high-throughput analysis of transcriptional regulation, including the functional characterization of transcription factors (TFs), cis-regulatory elements, and enhancer/silencer activity. This approach facilitates systematic promoter dissection through site-directed mutagenesis, deletion mapping, and combinatorial TF studies to uncover complex regulatory interactions. Here, we present a detailed protocol for performing dual-luciferase transient expression assays, emphasizing best practices for ensuring robust and reproducible results.