Abstract: Droplet microfluidics has made large impacts in diverse areas such as enzyme evolution, chemical product screening, polymer engineering, and single-cell analysis. However, while droplet reactions have become increasingly sophisticated, phenotyping droplets by a fluorescent signal and sorting them to isolate variants-of-interest remains a field-wide bottleneck. Here, we present an optimized double emulsion workflow, sdDE-FACS, that enables high-throughput phenotyping, selection, and sorting of droplets using standard flow cytometers. Using a 130 μm nozzle, we demonstrate robust post-sort recovery of intact droplets, with little to no shear-induced droplet breakage, at high sort frequency (12-14 kHz) across two instruments. We report the first quantitative plate statistics for droplet double emulsion isolation and demonstrate single droplet recovery with >70% efficiency. In addition, we establish complete downstream recovery of nucleic acids from single, sorted double emulsion droplets, comparable to the capabilities of single-cell FACS. This work resolves several hurdles in the field of high-throughput droplet analysis and paves the way for a variety of new droplet assays, including rare variant isolation and multiparameter single-cell analysis, marrying the full power of flow cytometry with droplet microfluidics. Manuscript files, device designs and details, and software are attached in this project. A full protocol for operating the sdDE-FACS can be found in the SI of our recent manuscript [here][2]. This protocol describes all the steps necessary to analyze and sort single double emulsion droplets via FACS at high-throughput. We also recommend use of our new workflow, Dropception, for encapsulating single cells in double emulsions and analyzing cell-containing droplets via FACS. A step-by-step protocol for this workflow can be found [here][1] in the SI accompanying the manuscript. Please feel free to email us with any and all questions! Contact: Kara Brower, kbrower@stanford.edu [1]: https://www.biorxiv.org/content/10.1101/2020.06.07.139311v1 [2]: https://pubs-rsc-org.stanford.idm.oclc.org/en/content/articlelanding/2020/lc/d0lc00261e#!divAbstract