The specific intracellular inhibition of protein activity at the protein level is a highly valuable tool for the validation or modulation of cellular processes. We demonstrate here the use of designed ankyrin repeat proteins (DARPins) as tailor-made intracellular proteinase inhibitors. Site-specific proteolytic processing plays a critical role in the regulation of many biological processes, ranging from basic cellular functions to the propagation of viruses. The NIa(pro) proteinase of tobacco etch virus, a major plant pathogen, can be functionally expressed in Escherichia coli without harming the bacterium. To identify inhibitors of this proteinase, we first selected binders to it from combinatorial libraries of DARPins and tested this pool with a novel in vivo screen for proteinase inhibition. For this purpose, a hybrid protein consisting of the omega subunit of E. coli RNA polymerase was covalently fused to a DNA-binding protein, the lambdacI repressor, containing an NIa(pro) cleavage site in the linker between the two proteins. Thus, this transcriptional activator is inactivated by site-specific proteolytic cleavage, and inhibitors of this cleavage can be identified by the reconstitution of transcription of a reporter gene. Following this two-step approach of selection and screening, we could rapidly isolate NIa(pro) proteinase inhibitors active inside the cell from highly diverse combinatorial DARPin libraries. These findings underline the great potential of DARPins for modulation of protein functionality in the intracellular space. In addition, our novel genetic screen can help to select and identify tailor-made proteinase inhibitors based on other protein scaffolds or even on low molecular weight compounds.