Proteases constitute currently about 5-10% of all pharmaceutical targets when looking for drugs against common diseases. In humans, proteases are a family of approximately 700 enzymes. Abnormal proteolytic activity is associated with cancer, diabetes, neurodegenerative diseases, respiratory diseases or bacterial and viral infections. One of the biggest problems in the investigation of proteases is very often their overlapping substrate specificity and location, but different function. This frequently limits discovery of specific molecules for selective imaging using available approaches. Currently, there are also not many selective chemical tools that can be reliably used to investigate these enzymes. To address this important problem, in this interdisciplinary project we plan to develop three completely new technologies based on unnatural and post-translationally modified amino acids, which should yield selective sequences in substrates and activity-based probes.