Abstract: The present invention relates to a genetically encoded FRET-based biosensor to monitor the activity of matrix metalloproteinase 9 (MMP-9). MMP-9 is an extracellular acting endopeptidase implicated in both physiological and pathological processes. A genetically encoded FRET biosensor anchored in the cellular membrane allows studying the proteolytic activity of MMP-9 with high spatiotemporal resolution at the exact region of MMP-9 action on the cell. Applicability of the biosensor, both in vitro and in vivo in living cells, has been demonstrated by ratiometric analysis of cleavage of the biosensor by a purified auto-activating mutant of MMP-9.