The ubiquitin-proteasome system plays a critical role in many diseases, making it an attractive biomarker and therapeutic target. However, the impact of results obtained in vitro using purified proteasome particles or whole cell extracts is limited by the lack of efficient methods to assess proteasome activity in living cells. We have engineered an internally quenched fluorogenic peptide with a proteasome-specific cleavage motif fused to TAT and linked to the fluorophores DABCYL and EDANS. This peptide penetrates cell membranes and is rapidly cleaved by the proteasomal chymotrypsin-like activity, generating a quantitative fluorescent reporter of in vivo proteasome activity as assessed by time-lapse or flow cytometry fluorescence analysis. This reporter is an innovative tool for monitoring proteasomal proteolytic activities in physiological and pathological conditions.