Over the last decade, new psychoactive substances (NPS) have been flooding the drug market. The high levels of harm associated with NPS use have urged many researchers to investigate their mechanism-of-action. Considering the ethical constraints in performing experiments on NPS in humans, there is much interest in the design of in vitro systems to study the signaling pathways of NPS leading to their effects in the human body. Here, we present a novel bioassay that quite literally helps casting light upon the pharmacology and toxicity of cannabinoids, opioids and psychedelics. By coupling the initiation of two distinct intracellular signaling pathways to the functional complementation of two luciferases, different colors of light will be emitted depending on the activated pathway. The second part of this project aims at optimizing the detection of NPS. The current screening methods (e.g. immunoassays) are often lagging behind as they are based on the detection of a specific structure or -for untargeted analysis- require sufficiently high analyte concentrations. Several activity-based assays have been developed to overcome these issues. However, for each type of NPS, a new assay is needed. Here, we describe a novel assay based on color-shifted nanoluciferases generating distinctly colored light upon activation of a specific GPCR, allowing multiplexing. Compared to performing separate assays, this system reduces workload and cost, rendering it very useful for screening purposes.