Matrix metalloproteinases (MMPs) are important players in skin homeostasis, wound repair, and in the pathogenesis of skin cancer. One member – MMP10 – is specifically expressed in wound keratinocytes and in epithelial cancer cells, but its function is unclear and epidermal substrates are poorly characterized. To unravel the role of MMP10 in the skin, we employed Terminal Amine Isotopic Labeling of Substrates (TAILS), an iTRAQ-based quantitative proteomics technique for the discovery of protease substrates and their cleavage sites, to monitor MMP10-dependent proteolysis over time in secretomes from keratinocytes. By time-resolved abundance clustering of neo-N termini we revealed a cleavage site specificity preference of MMP10 for glutamate in the P1 position and identified the α6 integrin subunit, cysteine-rich angiogenic inducer 61 and dermokine as novel MMP10 substrates. Moreover, we confirmed the same MMP10-dependent processing of dermokine in vivo by TAILS analysis of epidermis from transgenic mice that overexpress a constitutively active mutant of MMP10 in basal keratinocytes. Since these substrates have been associated with cell adhesion, differentiation and senescence, MMP10 might contribute to modulation of these processes during skin repair.