Single-molecule fluorescence resonant energy transfer (smFRET) allows identifying sub-populations within doubly-labeled molecules, based on the distances between the donor (D) and acceptor (A) fluorescent labels. Solution-based smFRET allows measurement of binding-unbinding events or conformational changes of dye-labeled biomolecules without ensemble averaging and free from surface perturbations. When employing dual (or multi) laser excitation, smFRET allows resolving the number of fluorescent labels on each molecule, greatly enhancing the ability to study heterogeneous samples. A major drawback to solution-based smFRET techniques is their low throughput, which renders measurements time-consuming and prevents from studying kinetic phenomena in real-time. Here we demonstrate a high-throughput smFRET setup which multiplexes acquisition by using 48 excitation spots and two 48 …