Matrix metalloproteinase (MMP)9 is a secreted protease, primarily functioning in the extracellular matrix. It is the most extensively glycosylated MMP, having two putative N-glycosylation sites in the prodomain and the catalytic domain and a heavily O-glycosylated linker domain. Importantly, also around 50% of known extracellular and membrane substrates of MMP9 are found to be either N- or O-glycosylated. However, the interplay of glycosylation and proteolytic processing of MMP9 substrates and its consequences for the modulation of biological activities have not been fully understood. Assessing the modulation of MMP9 proteolytic processing by substrate glycosylation through a combination of a multiplexed mass spectrometry-based substrate discovery approach, TMT-TAILS, and cellular engineering will allow systematically exploring the regulatory crosstalk between limited proteolysis and glycosylation in the extracellular space. Novel glycosylation-dependent processing events will be identified by a comparative assessment of MMP9 substrate cleavages in glycosylated and non-glycosylated cell secretomes, and the selected candidate substrates will be further validated and functionally characterized. This study will not only assess the fine-tuning of limited proteolysis in the extracellular space by glycosylation but also provide a better understanding of MMP9 regulation with strong implications for basic biology and translational approaches that aim at targeting aberrant protease activities in human diseases.