Developing selective inhibitors for proteolytic enzymes that share high sequence homology and structural similarity is important for achieving high target affinity and functional specificity. Here， we used a combination of yeast surface display and dual-color selective library screening to obtain selective inhibitors for each of the matrix metalloproteinases (MMPs) MMP14 and MMP9 by modifying the non-specific N-terminal domain of the tissue inhibitor of metalloproteinase-2 (N-TIMP2). We generated inhibitor variants with 30- to 1175-fold improved specificity to each of the proteases， respectively， relative to wild type N-TIMP2. These biochemical results accurately predicted the selectivity and specificity obtained in cell-based assays. In U87MG cells， the activation of MMP2 by MMP14 was inhibited by MMP14-selective blockers but not MMP9-specific inhibitors. Target specificity was also demonstrated in MCF-7 cells stably expressing either MMP14 or MMP9， with only the MMP14-specific inhibitors preventing the mobility of MMP14-expressing cells. Similarly， the mobility of MMP9-expressing cells was inhibited by the MMP9-specific inhibitors， yet was not altered by the MMP14-specific inhibitors. The strategy developed in this study for improving the specificity of an otherwise broad-spectrum inhibitor will likely enhance our understanding of the basis for target specificity of inhibitors to proteolytic enzymes， in general， and to MMPs， in particular. We， moreover， envision that this study could serve as a platform for the development of next-generation， target-specific therapeutic agents. Finally， our methodology can be extended to other classes of proteolytic enzymes and other important target proteins.