CMAC (7-amino-4-chloromethylcoumarin) staining is a valuable tool for visualizing acidic organelles, including vacuoles, in yeast cells. By selectively accumulating in acidic compartments, CMAC allows specific labeling and visualization of vacuoles in living or fixed cells, aiding in the investigation of vacuole dynamics, morphology, and distribution under various conditions. This staining method provides a clear contrast against cellular autofluorescence, enhancing imaging clarity. It can be combined with automated imaging capture for faster throughput and analysis. Characterizing vacuole phenotypes using CMAC staining contributes to our understanding of cellular homeostasis and disease mechanisms in yeast. It has applications in studying fundamental cellular processes, including endocytic trafficking and autophagy, as well as in drug screening assays, and can be used in yeast models of disease to offer insights into potential therapeutic targets for diseases affecting the lysosome, including neurodegenerative diseases.