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Single molecule FRET (smFRET) is a powerful tool for looking at protein folding and conformational dynamics (1). The application of smFRET to interesting proteins involved in pathologies or drug targeting is limited when the protein of interest is difficult to express in vivo due to toxicity and where cell-free expression is required. Moreover, site-specific labeling is an important issue for smFRET (2), but there is still a lack in simple and robust methods for site-specific labeling of in vitro synthesized proteins for smFRET studies. The conventional method is to double label the protein via cysteine residues (3), but as a consequence of a single chemistry, labeling specificity is limited and would lead to an inaccurate picture of the studied population (4). Incorporation of unnatural amino acids carrying an orthogonal chemistry can increase labeling specificity as it was reported (4). However, described methods were so far mainly considered for smFRET studies performed on in vivo synthesized proteins.By using human calmodulin (hCaM) as a model protein we have developed an alternative method for site-specific labeling of cell-free synthesized proteins. Site-specificity of our system allowed us to obtain sharper FRET histograms compared to the conventional labeling method. In addition, by demonstrating functionality of the labeled hCaM through binding experiments performed in presence of ligands and partners, we have shown the biological relevance of our method.In summary, we have developed a robust and simple method that enables the accurate study of cell-free synthesized proteins in smFRET. This method can be used for studying proteins that cannot be expressed in vivo, as it would be for toxic or membrane proteins.1. Tan, Y. W., J. A. Hanson, J. W. Chu, and H. Yang. 2014. Confocal single-molecule FRET for protein conformational dynamics. Methods Mol. Biol. 1084:51-62.2. Joo, C., and T. Ha. 2012. Labeling proteins for single-molecule FRET. Cold Spring Harb. Protoc. 2012:1009-1012.3. Kim, Y., S. O. Ho, N. R. Gassman, Y. Korlann, E. V. Landorf, F. R. Collart, and S. Weiss. 2008. Efficient site-specific labeling of proteins via cysteines. Bioconjug. Chem. 19:786-791.4. Seo, M. H., T. S. Lee, E. Kim, Y. L. Cho, H. S. Park, T. Y. Yoon, and H. S. Kim. 2011. Efficient single-molecule fluorescence resonance energy transfer analysis by site-specific dual-labeling of protein using an unnatural amino acid. Anal. Chem. 83:8849-8854.