Lysosomes are membrane-bound subcellular organelles involved in the degradation of macromolecules and pathogens in diverse processes, including endocytosis, phagocytosis, and autophagy. A red fluorescent probe was developed that is selectively sequestered in acidic organelles. U20S cells pretreated with 64 microM chloroquine for as little as 5 h show a dramatic increase in lysosome-like vesicle number and volume. The probe can be employed for highlighting lysosome-like organelles under conditions wherein cells produce vacuoles that contain most of the degradative enzymes of the lysosome but are not as acidic as the parent organelle. Using a conventional fluorescence microplate reader, the half-maximal effective concentration (EC(50)) of chloroquine was estimated. The high Z' score obtained using the assay demonstrated excellent signal-to-noise ratios. The fluorescence microplate assay was successfully employed to screen a small-molecule compound library for agents that increase lysosomal volume and number. One potential application of the new assay is in the toxicology portion of preclinical drug safety assessment (ADME-Tox) workflows, using in vitro cell culture models to aid in the drug development process.