Transcription termination mediated by the ring-shaped, ATP-dependent Rho motor is a multipurpose regulatory mechanism specific to bacteria and constitutes an interesting target for the development of new antibiotics. Although Rho-dependent termination can punctuate gene expression or contribute to the protection of the genome at hundreds of sites within a given bacterium, its exact perimeter and site- or species-specific features remain insufficiently characterized. New advanced approaches are required to explore thoroughly the diversity of Rho-dependent terminators and the complexity of associated mechanisms. Current in vitro analyses of Rho-dependent termination rely on radiolabeling, gel electrophoresis, and phosphorimaging of transcription reaction products and are thus hazardous, inconvenient, and low-throughput. To address these limitations, we have developed the first in vitro assay using a fluorescence detection modality to study Rho-dependent transcription termination. This powerful experimental tool accurately estimates terminator strengths in a matter of minutes and is optimized for a microplate reader format allowing multiplexed characterization of putative terminator sequences and mechanisms or high-throughput screening of new drugs targeting Rho-dependent termination.