Multiplexed cellular screens offer a powerful solution to the limitations of traditional, single-target assessment-based experiments by allowing scientists to measure multiple biological functions simultaneously. For past several decades, dual luciferase reporter assay using non-overlapping firefly and Renillaluciferase formed the backbone of biomedical and biotechnology research applications. Although it was highly reliable, the application could essentially allow measurement ofonly one gene function assessment at one go. Our recent report of ‘triple reporter assay’ development by integrating the mushroom luciferase (nnLuz) isolated from Neonothopanus nambihas renewed the scope of developing a non-overlapping, multiplexed reporter assay for simultaneous measurement of multiplegene functions from live cells. This development adds up strength to biomedical research applications with requirement of large volume screens for modulators against the function of multigene complexes, which are relevant in the context of normal or disease biology.