Research and diagnostic tools for high-throughput molecular detection can typically target only a single analyte or class of analytes at a time. One approach to addressing this shortcoming is suspending cell-free expression (CFE) detection systems in aqueous two-phase system (ATPS) droplets, then spatially assembling different protocell droplets into an assay capable of detecting diverse analytes. However, this approach necessitates costly preparation of cell-free lysates. Thus, we developed E.CoDROP, a novel method for rapidly creating cell extracts within ATPS droplets. To create these droplets, Escherichia coli cells grow to exponential growth phase, pellet and resuspend in Ficoll (10 w/v%), and vortex with 35k PEG (5 w/v%). Within the resultant ATPS droplets, cells lyse, caused by a gene cassette under control of an arabinose-inducible pBAD system. Lysis releases cell contents, including transcription-translation machinery and any plasmid-encoded molecular detection systems. These droplets are then suitable for use in the aforementioned CFE detection systems.