Lysosomes are membrane-bound organelles for biomolecule degradation and recycling. They also serve as a nutrient sensing and signaling center to maintain cell and tissue homeostasis. Lysosomal properties alter in response to developmental or environmental cues, but these changes are hard to track in vivo. Employing C. elegans as a model system, we have developed assays to examine and quantify lysosome properties in vivo, including lysosome maturation, acidification and cleavage activity. These assays can be used to reveal alterations of lysosomal activity during C. elegans development and in stress conditions.