Bacteria can exhibit phenotypes that render them tolerant against antibiotics. However, often only a few cells of a bacterial population show the so-called persister phenotype, which makes it difficult to study this health-threatening phenotype. We recently found that certain abrupt nutrient shifts generate Escherichia coli populations that consist almost entirely of antibiotic-tolerant cells. These nearly homogeneous persister cell populations enable assessment with population-averaging experimental methods, such as high-throughput methods. In this chapter, we provide a detailed protocol for generating a large fraction of tolerant cells using the nutrient-switch approach. Furthermore, we describe how to determine the fraction of cells that enter the tolerant state upon a sudden nutrient shift and we provide a new way to assess antibiotic tolerance using flow cytometry. We envision that these methods will facilitate research into the important and exciting phenotype of bacterial persister cells.