Cockroach and Alternaria alternata are common allergens to which sensitization is associated with the development of asthma. Trypsin-like activity that activates Proteinase Activated Receptor-2 (PAR2) has been reported in both allergens, though the cockroach- and Alternaria-derived proteinases have yet to be identified and characterized. Trypsin-specific activity-based probe labeling revealed three distinct enzymes in the cockroach allergen (E1, E2, E3), and one in the Alternaria allergen (Alt). Each of the enzymes was isolated and purified with ion-exchange chromatography, and the isolated enzymes characterized by determining their biochemical properties. The enzymes were distinct based on their: (1) selectivities for fluorogenic peptide substrates (Km), (2) sensitivities to serine proteinase inhibitors (Ki), and (3) amino acid sequences (mass spectrometral sequencing). Each enzyme was also assessed for its ability to activate/dis-arm PAR1 and PAR2 by (1) measuring Ca2+ signaling, (2) measuring MAPK phosphorylation, and (3) monitoring the cleavage of PAR-linked luciferase constructs. Each of the enzymes was found to activate PAR2-mediated Ca2+ and MAPK signaling at equal concentrations. The enzymes were found to regulate PAR1 differentially; cockroach E1 and E3 are biased agonists for PAR1, preferentially activating MAPK signaling over Ca2+ signaling, whereas E2 and Alt are non-biased agonists for PAR1, activating both Ca2+ and MAPK signaling at relatively low concentrations. Finally, the ability of the allergen-derived enzymes to regulate PAR signaling in airway-derived epithelial cells and mouse bronchial tissues was assessed. Each allergen enzyme activated MAPK signaling in the airway cells, and activation of the cells with E1 and Alt induced IL-8 expression. Enzymatic regulation of PAR signaling at the tissue level was assessed as the smooth muscle relaxation response in mouse aortic and bronchial tissues. Alt and E1 were able to induce relaxation via PAR2 in tissues from PAR1 knockout mice, but only Alt elicited a relaxation response via PAR1 in PAR2-null tissue. PAR signaling in the airway has been shown to contribute to inflammation and allergic sensitization, so each of the four allergen-derived trypsin-like enzymes are potential therapeutic targets in allergic airway disease.