Human β-tryptase, a trypsin-like serine protease that is active only as a tetramer, is an important mediator of allergic inflammatory responses in asthma. It is present in the secretory granules of mast cells and released upon mast cell activation as a complex with proteoglycans that stabilize the tetramer. Severe asthma patients with low type 2 inflammation derive less clinical benefit from therapies targeting type 2 cytokines, and represent an unmet clinical need. We show that mast cell tryptase is elevated in severe asthma patients independent of type 2 biomarker status. Active β-tryptase allele count correlates with blood tryptase levels, and asthma patients carrying more active alleles benefit less from anti-IgE treatment. While small molecule inhibitors that target the active site of serine proteases have been developed as drugs, there are significant challenges to find compounds with high specificity. Antibodies are very selective and generally inhibit proteases by blocking substrate access by binding to active sites or exosites or by allosteric modulation. We generated various inhibitory antibodies against human β-tryptase, all of which allosterically dissociate active tetramers into inactive monomers. However, these antibodies all have quite distinct molecular mechanisms of action. Structural and biochemical studies including EM, X-ray and HDX of various antibodies reveal the molecular basis for allosteric destabilization of small and/or large interfaces required for tetramerization. Both monovalent and bivalent-dependent mechanisms have been found. Notably, inhibition of β-tryptase activity by the bivalent-dependent IgG does so in a hinge-dependent manner, acting as a molecular pliers to dissociate the tetramer. We show that an anti-tryptase antibody potently blocks tryptase enzymatic activity in a humanized mouse model, reducing IgE-mediated systemic anaphylaxis and inhibits airway tryptase in Ascaris-sensitized cynomolgus monkeys with favorable pharmacokinetics. These data provide a foundation for developing anti-tryptase as a clinical therapy for severe asthma, particularly for type 2 inflammation-independent subsets.