Luciferase genes have been used extensively for quantitative analysis in RNA interference (RNAi) and endogenous microRNA (miRNA) studies. However, one drawback is that determination of luciferase activity always requires that cells be killed, allowing less real-time information about a biological process to be obtained. Here we describe a triple-reporter plasmid for target miRNA analysis in which enhanced green fluorescent protein (EGFP) and Renilla luciferase (RLuci) are linked by "self-cleave" 2A under control of the CMV promoter. Firefly luciferase (FLuci) serves as internal control under control of another independent promoter. Our real-time system provides a convenient and improved approach for assessing messenger RNA silencing in vivo.