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Förster resonance energy transfer (FRET) studies performed at the single molecule level have unique abilities to probe molecular structure, dynamics, and function of biological molecules. This technique requires specimens, like proteins, equipped with two different fluorescent probes attached at specific positions within the molecule of interest. Here, we present an approach of cell-free protein synthesis (CFPS) that provides proteins with two different functional groups for post-translational labeling at the specific amino acid positions. Besides the sulfhydryl group of a cysteine, we make use of an azido group of a p-azido-l-phenylalanine to achieve chemical orthogonality. Herein, we achieve not only a site-specific but, most importantly, also a site-selective, label scheme that permits the highest accuracy of measured data. This is demonstrated in a case study, where we synthesize human calmodulin (CaM) by using a CFPS kit and prove the structural integrity and the full functionality of this protein.