Characteristics of fluorogenic substrates designed for detection of enzyme activity in living cells are reviewed. Improved retention of the fluorescent products in the cell of origin can be achieved by structural modifications to the substrate that result in association with membrane lipids or conjugation to intracellular glutathione. Newly-developed substrates that yield fluorescent precipitates provide the additional advantage of allowing subcellular localization of sites of enzymatic activity. Improved detection sensitivity can also be achieved by targeted delivery of substrates for processing by specific organelles. Substrates designed for monitoring oxidative activity and lipid metabolism provide examples of this approach.