Specific enzymes play key roles in many pathophysiological processes and therefore are targets for therapeutic strategies. The activity of most enzymes is largely determined by many factors at the post-translational level. Therefore, it is essential to study the activity of target enzymes in living cells and tissues in a quantitative manner in relation to pathophysiological processes to understand its relevance and the potential impact of its targeting by drugs. Proteases, in particular, are crucial in every aspect of life and death of an organism and are therefore important targets. Enzyme activity in living cells can be studied with various tools. These can be endogenous fluorescent metabolites or synthetic chromogenic or fluorogenic substrates. The use of endogenous metabolites is rather limited and nonspecific because they are involved in many biological processes, but novel chromogenic and fluorogenic substrates have been developed to monitor activity of enzymes, and particularly proteases, in living cells and tissues. This review discusses these substrates and the methods in which they are applied, as well as their advantages and disadvantages for metabolic mapping in living cells.