he advance of robotic liquid handling platforms and ever more efficient experimental procedures allowed high-throughput analysis of many biological samples. However, the growing need to analyze large numbers of single-cells sets further constrains. Droplet microfluidics provides a unique opportunity to overcome these obstacles as it allows sensitive high-throughput analysis of large heterogeneous cell populations, at a fraction of the usual cost and time. Herein we present a novel FRET-based droplet microfluidic assay for the screening and sorting of single cells secreting monoclonal antibodies (mAbs) of desired specificity. Single cells are encapsulated into 40 pL droplets along with two fluorescently labeled probe molecules. Only in droplets containing the secreted mAb of interest, the FRET reaction between the two probe molecules occurs, leading to the emission of a specific fluorescence signal. The signal is subsequently detected and used as trigger for on-chip dielectrophoretic sorting. Single cells are analyzed and sorted at a rate of approximately 500 Hz, with efficiency exceeding 98%. Our FRET assay overcomes the disadvantages of many previously published microfluidic detection systems, relying on inefficient co-encapsulation events. Moreover, it overcomes the key limitation of conventional fluorescence activated droplet sorting (FACS), by entrapping the secreted mAbs and the cells that produce them in droplets. The presented platform should be appealing for the analysis of secreted molecules by heterogeneous cell populations.