The export of viral RNA from the nucleus to the cytoplasm of the cellular host is a crucial step in the life cycle of HIV-1 that is mediated by the viral Rev protein. One aspect of the Rev function, its multimerization, is still unexplored as a target for antiviral therapy. This is partly due to the lack of a fast and solid system to measure Rev multimerization. We have developed a high throughput in vitro Rev multimerization assay based on fluorescence resonance energy transfer (FRET) in which real-time Rev-Rev interactions can be measured both in the absence and the presence of Rev specific RRE RNA. Well-characterized Rev multimerization deficient mutants showed reduced FRET as well as unlabeled Rev molecules were able to inhibit the FRET signal demonstrating the specificity of the assay. Upon multimerization along RRE RNA the FRET signal significantly increased but dropped again at equimolar Rev/RRE ratios suggesting that in this condition most Rev molecules are bound as monomers to the RRE. Furthermore, using this assay, we demonstrate that a previously selected llama heavy-chain only antibody was shown to not only prevent the development of Rev multimers but also disassemble the already formed complexes confirming the dynamic nature of the Rev-Rev interactions. The in vitro FRET based multimerization assay facilitates the further study of the basic mechanism of cooperative Rev multimerization along the RRE and is also widely applicable to study the assembly of other functional complexes involving protein homo-multimerization or cooperative protein-protein interactions on RNA or DNA.