Here we report the design, chemical and recombinant synthesis, and functional properties of a series of novel inhibitors of human mast cell tryptase beta, a protease of considerable interest as a therapeutic target for the treatment of allergic asthma and inflammatory disorders. These inhibitors are derived from a linear variant of the cyclic cystine knot miniprotein MCoTI-II, originally isolated from the seeds of Momordica cochinchinensis. A synthetic cyclic miniprotein that bears additional positive charge in the loop connecting the N- and C-termini inhibits all monomers of the tryptase beta tetramer with an overall equilibrium dissociation constant K(i) of 1 nM and thus is one of the most potent proteinaceous inhibitors of tryptase beta described to date. These cystine knot miniproteins may therefore become valuable scaffolds for the design of a new generation of tryptase inhibitors.