The proteasome is an important component of the intracellular system for the turnover of proteins. The mammalian proteasome is engaged to degrade a bulky fraction of soluble intracellular proteins both in an ubiquitin-dependent and independent manner. The proteasome is composed by a central catalytic core--the 20S proteasome--where three different proteases are located, whose activities can be measured. A detailed protocol for measuring accurately the three activities of the 20S proteasome in cell and tissue homogenates, using specific fluorogenic substrates and a microplate reader fluorometer, are described. Successful applications of this method include determining changes in the proteasomal activities during aging, anti-aging interventions, cell cycle analysis, and in various disease states including neurodegenerative diseases and cancers.