Hepatitis C is a liver disease caused by hepatitis C virus (HCV) infection. Protease inhibitor (PI) is included in the current oral direct-acting antiviral (DAA) combination therapy. However, these HCV PIs are small molecule drugs; therefore, they could have serious off-target adverse effects. New types of treatment such as antibody drugs that have very high specificity and selectivity would be a better alternative. Our study reports the application of the PROTECT (Protease inhibitor Recognition based On Tat Export after Cleavage Tampering) assay, an in vivo detection method for protease inhibiting intracellular antibodies (intrabodies) based on the bacterial twin-arginine translocation (Tat) pathway to the HCV NS3 protease system. This assay was designed such that when protease is co-expressed inside the bacterial cytoplasm, Tat transportation of the uncleaved protease substrate due to protection of the cleavage site by a specific intrabody will result in β-lactam antibiotic resistance. Using the anti-NS3 intrabodies isolated previously, we demonstrated that PROTECT assay could distinguish between the inhibitory and non-inhibitory anti-NS3 intrabodies resulting in selective growth in the presence of β-lactam antibiotics. This method has potential for the screening of agents that inhibit proteolytic cleavage in a bacterial cell-based assay, which may find use in identifying, reconstituting, and characterizing protease inhibitors, identifying mutations on the substrate that can inhibit proteolytic cleavage, and drug screening.