Human Argonaute 2 (hAgo2) is the key player of RNA interference (RNAi), a posttranscriptional mechanism that regulates a major portion of human genes, including a high number of genes involved in disease-related processes. To exert its function, hAgo2 binds short RNAs that guide the enzyme to its cognate mRNAs target via base complementarity between guide and target. Incomplete complementarity between miRNA guides and target mRNA leads to the recruitment of additional proteins that promote mRNA decay or translational inhibition of the bound mRNA. Alternatively, direct cleavage of the bound mRNA by hAgo2 is possible. As common to transient protein-nucleic acids complexes, interactions have to be established and also be disrupted to allow substrate and interaction partner exchange during hAgo’s activity cycle. This demands a certain degree of structural flexibility in hAgo2. X-ray crystal structures provided valuable information about the stable conformations that hAgo2 adopts upon guide and target RNA binding. However, the dynamic aspect of hAgo2 action could not be elucidated as hAgo2 is not amenable to recombinant production thereby preventing conventional site-specific protein labelling schemes necessary for FRET measurements. In order to solve this problem and to use the native state of hAgo2 including all necessary post-translational modifications like e.g. phosphorylation, we developed the SLAM-FRET (Site-specific labelling of endogenous mammalian proteins for single-molecule FRET measurements) workflow (1). Making use of this method, we were able to conduct smFRET measurements with site-specifically fluorescently labeled hAgo2 (carrying all posttranslational modifications) and/or labeled guide and target RNAs or additional interaction partners. This way, we observed the conformational evolution of hAgo2 throughout its activity cycle. Among others, we found that hAgo2 can adopt an open and closed conformation and a dynamic switching between conformations occurs. Moreover, interaction partners like TNRC6 appear to modulate the conformation of hAgo2. Hence, our data complement the structural information on hAgo2 and provide insights into the mode of interaction between hAgo2 and additional proteins that are part of the RNAi pathway.