Enzymes, especially proteases, play critical roles in almost every aspect of biological and pathological processes. The abnormal activation or inhibition of protease activity will result in serious human diseases such as peritonitis, atherosclerosis and cancer. There is a strong need to develop protease sensors capable of quantitatively measuring the protease activity in real time and monitoring protease activation and inhibition in various cellular compartments. In this chapter, we present a straightforward approach to monitoring protease activity in real time in live cells with protease sensors developed in our lab. These protease sensors have been developed based on a single enhanced green fluorescent protein (EGFP) with a larger ratiometric change of fluorescence signal after cleavage by protease, such as trypsin and caspase-3. The activation of these proteases are captured by real-time fluorescence microscopy. This novel approached can be further applied to probe disease mechanisms corresponding to proteases activities and to screen protease inhibitors with therapeutic effects in cells.