A synthetic signaling platform has been developed featuring proteases as elementary signal transducing units. Individual signal transducers are highly engineerable featuring a high degree of orthogonality and modularity as viral proteases with stringent and well-defined substrate specificities are recombined with engineered inhibitors and different types of sensor moieties in a plug-and-play fashion. Notably, with just two artificially autoinhibited protease modules based on proteases derived from HCV and TVMV, it was possible to create a range of protease-based molecular switches and signal sensing motives. For instance, highly specific protease biosensors were constructed by introducing cleavage sites for different clinically important proteases (e.g. thrombin and prostate specific antigen) into the sensor moiety while the sensitivity of individual biosensors could be enhanced by means of affinity targeting using peptides and antibody-like fragments that tightly bind their target proteases. Moreover, protease-based molecular switches have been developed that can sense the presence of a small molecule ligand and translate it into a proteolytic signal. In one format, an artificially autoinhibited version of HCV is selectively activated following rapamycin induced co-localization of TVMV in both constitutively active and autoinhibited forms. In a second format, an allosterically regulated protease was engineered by connecting TVMV to its inhibitor via an ‘affinity clamp’, an artificially engineered receptor scaffold that undergoes a large conformational change upon ligand binding. The receptor displays an unprecedented functional plasticity as its responsiveness upon ligand binding can be configured from 4-fold switch-OFF to 60-fold switch-ON solely by varying the length of the linkers that connect the ‘affinity clamp’ with TVMV and its inhibitor. Using a second autoinhibited transducer protease based on HCV, it is possible to form a linear amplification cascade and amplify the signal with improved sensitivity. Overall, we anticipate this signaling platform to find widespread applications in diagnostics, and will also allow us to probe fundamental principles that underlie biomolecular signaling events using defined components in vitro and in live cells.