The high-throughput analysis and isolation of bacterial cells encapsulated in agarose microparticles using fluorescence-activated cell sorting (FACS) is described. Flow-focusing microfluidic systems were used to create monodisperse microparticles that were ∼30 μm in diameter. The dimensions of these particles made them compatible with flow cytometry and FACS, and the sensitivity of these techniques reduced the incubation time for cell replication before analyses were carried out. The small volume of the microparticles (∼1-50 pL) minimized the quantity of reagents needed for bacterial studies. This platform made it possible to screen and isolate bacteria and apply a combination of techniques to rapidly determine the target of biologically active small molecules. As a pilot study, Escherichia coli cells were encapsulated in agarose microparticles, incubated in the presence of varying concentrations of rifampicin, and analyzed using FACS. The minimum inhibitory concentration of rifampicin was determined, and spontaneous mutants that had developed resistance to the antibiotic were isolated via FACS and characterized by DNA sequencing. The β-subunit of RNA polymerase, RpoB, was confirmed as the target of rifampicin, and Q513L was the mutation most frequently observed. Using this approach, the time and quantity of antibiotics required for the isolation of mutants was reduced by 8- and 150-fold, respectively, compared to conventional microbiological techniques using nutrient agar plates. We envision that this technique will have an important impact on research in chemical biology, natural products chemistry, and the discovery and characterization of biologically active secondary metabolites.