Since protease activity is highly regulated by structural and environmental influences, the abundance of a protease often does not directly correlate with its activity. Because in most of the cases it is the activity of a protease that gives rise to its biological relevance, tools to report on this activity are of great value to the research community. Activity-based probes (ABPs) are small molecule tools that allow for the monitoring and profiling of protease activities in complex biological systems. The class of fluorescent quenched ABPs (qABPs), being intrinsically “dark” and only emitting fluorescence after reaction with the target protease, are ideally suited for imaging techniques such as small animal noninvasive fluorescence imaging and live cell fl uorescence microscopy. An additional powerful characteristic of qABPs is their covalent and irreversible modification of the labeled protease, enabling in-depth target characterization. Here we describe the synthesis of a pan-cysteine cathepsin qABP BMV109 and the application of this probe to live cell fl uorescence imaging and fluorescent SDS-PAGE cysteine cathepsin activity profiling.