ABSTRACTDue to rising antibiotic resistance, there is an urgent need for different treatment options for multidrug-resistant infections. One alternative under investigation is phage therapy, which uses phages to treat bacterial infections. Although phages are highly abundant in the environment, not all phages are suitable for phage therapy, and finding efficient phages that lack undesirable traits such as bacterial virulence factors is challenging. Here, we developed a targeted single-phage isolation method to detect and isolate phages of interest and to characterize their kinetics in a high-throughput manner. This assay has also revealed cell-to-cell variations at a single-cell level among cells infected with the same phage species, as well as among cells infected with different phage species.IMPORTANCEThe spread of multidrug-resistant bacteria is a global human health threat, and without immediate action we are fast approaching a postantibiotic era. One possible alternative to antibiotics is the use of phages, that is, bacterial viruses. However, the isolation of phages that effectively kill their target bacteria has proven challenging. In addition, isolated phages must go through significant characterization before their efficacy is measured. The method developed in this work can isolate single phage particles on the basis of their similarity to previously characterized phages while excluding those with known undesirable traits, such as bacterial toxins, as well as characterizing their kinetics. Using this method, we revealed significant cell-to-cell variations in phage kinetics at a single-cell level among highly virulent phages. These results shed some light on unknown phage-bacterium interactions at the single-cell level.