The yeast proteome includes about 300 polytopic membrane proteins known or predicted to function as transporters. Such proteins ensure active or passive transport of small ions or metabolites across the plasma or internal membranes. Despite decades of research on yeast transporters, many of these remain uncharacterized in terms of substrate selectivity range, subcellular localization, and biological function. Assaying the uptake of radiolabeled compounds into whole cells or isolated organelles remains a powerful method for characterizing the function and biochemical properties of these proteins. Here we describe established protocols for measuring transporter activity in whole cells, intact vacuoles, or reconstituted vacuolar vesicles. These methods have proved particularly useful in the context of our work on yeast amino acid transporters, and can in principle be applied to assaying the uptake of other categories of compounds.