Hydrolases are enzymes (i.e. proteases, esterases, lipases) that often play pathogenic roles in many common human diseases such as cancer, asthma, arthritis, atherosclerosis and infection by pathogens. Therefore, tools that allow dynamic monitoring of their activity can be used as diagnostics, as imaging contrast agents and for the identification of novel enzymes as drug leads. In this presentation, I will describe our efforts to design and build libraries of privileged scaffolds containing reactive electrophiles to identify covalently binding lead molecules that can be converted into selective inhibitors as well as fluorescent active site probes for imaging hydrolase activity in vivo. This will include recent advances using fluorescent probes for real-time visualization of tumors during surgery as well our efforts to identify several new classes of serine hydrolases in pathogenic and commensal bacteria. The presentation will also focus on our efforts to expand our libraries of electrophiles using modern solid phase synthesis methods.