Analysis of multiple signaling pathways in the same cells at the same time requires orthogonal genetically encoded reporters with large dynamic ranges. Luciferases have arisen as cost-effective, versatile candidates whose output signals can be sensitively detected. Commonly used dual luciferase reporter assays detect one luciferase that is coupled to a single cellular pathway, and a second that is coupled to a control pathway for normalization purposes. We recently expanded this approach towards multiplex hextuple luciferase assaying that can report on five cellular signaling pathways and one control, each of which is encoded by a unique luciferase. Light emission by the six luciferases can be distinguished by the use of distinct substrates and by decomposing the emitted light using the emission spectra of each enzyme. Using an adaptable synthetic assembly cloning pipeline that can be applied to any cellular pathways, all six luciferase reporter units are stitched together into a single vector. Through use of a single vector that delivers stoichiometric ratios of each multiplex element to each transfected cell, this approach proposes to step away from traditional cotransfection methods typically used for luciferase or any other reporter assays, using a method we refer to as solotransfection. As a proof of concept, we engineered a luciferase assay tailored to probe pathway fluxes through c-Myc, NF-κβ, TGF-β, p53, and MAPK/JNK transcriptional response elements against the control constitutive CMV promoter. Using existing luciferase reagents and a variety of cell lines, we were able to monitor the effects of siRNA, ligand, and chemical compound treatments on their target pathways and on the four other cellular pathways at the same time. Currently, we are expanding the number of pathway reporters that can be stitched together as needed and exploring additional applications for multiplex luciferase assaying. Our work demonstrates that multiplex luciferase assays have broad application potential across different research fields to monitor pathways simultaneously.