Furthermore, we were able to demonstrate kinetic characterization of the solubilized receptor using FCS. For comparison, in a recent publication describing the cell-free synthesis of functional adrenergic receptor b2 complexed with nanodiscs, the receptor required insertion of a T4 lysozyme sequence in the loop region to obtain functional adrenergic receptor b2 protein. Using our method NK1R, ADRB2 and DRD1 were all functional in ligand binding GW786034 assays after a single-step co-expression and co-assembly system without requiring detergents or protein modification for stabilization. It is also worth noting that in other nanodisc-related GPCR studies or cell-free production of GPCR assays, separate protein production and purification preprocessing with detergents was required prior to NLP complex assembly. Our results indicate that adding additional purification steps can be avoided as well as the requirement for using a fusion protein for stabilizing the GPCRs. Assessment of NK1R activity was independently validated by three different methods that included fluorescent dot blot assays, EPR spectroscopy and FCS. Dot blot assays and EPR spectroscopy demonstrated that NK1R loaded into NLPs were bioactive. Furthermore, the nM affinities were comparable to earlier published studies using mammalian derived NK1R. Among these three approaches, FCS is a particularly powerful tool for characterizing NLPs, as it provided a more quantitative approach to rapidly determine the solution-based binding constants for NK1R-SP interaction studies. FCS also enabled us to determine the hydrodynamic radii of the diffusing complexes along with their concentrations. This can be overcome by an appropriate design of a combinatorial screen of initial concentrations for NK1R-NLPs and SP. Mixing fluorescently labeled compounds with appropriate amounts of unlabeled compounds is the strategy for extending the concentration range. After reaching equilibrium, the actual concentrations of each species were then inferred and used to calculate the dissociation constant. The most popular method for screening binding activity for GPCRs is using radioactivity assays, however this is often disadvantageous since it requires the handling of isotope labeled ligands. Other screening approaches include dot blot assays and EPR spectroscopy as described above. All of these methods require larger amounts of reagents that are not always easily achievable for the GPCRs of interest. In comparison, FCS can be performed in small volumes or even less when microfluidic delivery methods are employed, and it is very sensitive to concentrations as in the range as low as picomolar. Therefore, generalizing the method of using FCS to assess binding constants for screening other GPCRs is warranted. Lastly, FCS can be extended to become a high-throughput cellfree screening platform for GPCRs by facilitating simultaneous measurements in multi-well plates, providing real-time monitoring of production, purification, and functionality of GPCRs as well as other synthetic receptors based on the cross correlation between signals from the proteins and their specific ligands as demonstrated here. Furthermore, the diffusion curves provide detailed structural information about the particular association between GPCRs and NLPs to form complexes as well as monitoring interactions between GPCRs and specific ligands or other small molecules such as lipids. In contrast to cell-based assays, our approach is currently limited to demonstrating ligand binding for a very specific set of GPCRs.