In combination with Gleason grade is predictive of recurrence of prostate cancer and that its clinical application might avoid unnecessary aggressive interventions, thus improving quality of life and reducing healthcare related expenses. Cell-free protein production has become a widely accepted means to speed up the production and characterization of this class of membrane proteins, as over-expression of membrane proteins in vivo typically results in cell toxicity, protein aggregation, misfolding, and low yield. Cell-free expression can also alleviate problems such as the need for time-consuming cloning, cell transfection, cell growth, cell lysis, and challenges related to subsequent purification. Cell-free systems permit unique labeling or tagging strategies not always readily available to whole cell systems for protein characterization. They have also proven beneficial to MK-2206 1032349-77-1 structural studies by NMR and X-ray crystallography. However, previously published studies of cell-free production of GPCRs typically required expression and subsequent purification combined with detergent solubilization of the proteins. This often alters the conformation and function of these membrane proteins. To overcome these aforementioned problems, functional membrane proteins can be assembled in lipid/protein-based particulate structures connoted as “nano-discs”, or nanolipoprotein particles. Such methods were previously used to express GPCRs and model proteins such as bacteriorhodopsin reconstituted into NLPs. These nanoparticle complexes form a compelling approach for the stabilization and characterization of membrane proteins. Recent work has described the self-assembly of a single integral membrane protein into soluble nanoparticulate phospholipid bilayers. This approach has previously been applied to the adrenergic receptor b2, and rhodopsin reconstituted into NLP constructs and shown to efficiently activate the associated G protein. This process, however, relied on the separate expression and detergent extraction of GPCRs of interest.. We have previously demonstrated a single-step cell-free approach for the expression of nanodisc-associated bacteriorhodopsin, a 7 transmembrane spanning protein, which is the structural model protein for rhodopsins and other GPCR family members. This complex was characterized using fluorescence correlation spectroscopy and showed unique diffusion behaviour in solution. Single molecule fluorescence techniques have been used to study GPCR interactions in vivo since the early 2000s as well as to NLP associated complexes. These earlier studies set the stage for further development of FCS to address protein-protein associations combined with kinetic characterization of GPCRs. In this paper we report the de novo synthesis of several active human GPCRs, and rapid solution-based functional binding studies using FCS, a single molecule fluorescence technique. Electron paramagnetic resonance spectroscopy and fluorescent dot blot assays were used for comparison as well. Compared to the other assays, FCS provided a more quantitative approach to rapidly determine the solution-based binding constants for GPCR-ligand interactions. FCS was advantageous by requiring small volumes of material for kinetic assessment. Moreover, FCS can be extended to become a high-throughput cell-free screening platform for GPCRs. Compared to other approaches for obtaining membrane-bound receptor proteins, cell-free co-expression provides a one-step viable method to produce functional GPCRs such as NK1R.