We have previously generated a semi-synthetic single-chain variable fragments (scFv) phage display library built on a thermodynamically stable single-framework scaffold. All scFv antibodies selected from this repertoire showed high thermodynamic stability and were expressed as soluble molecules in bacterial cytoplasm. In this work, two complementary methodologies have been adopted to assess the functionality of library-derived scFvs as intracellular antibodies and to verify the possibility to directly use this repertoire for the selection of antibodies able to function in a reducing environment. The possibility to improve the performance of this highly stable antibody repertoire was evaluated subjecting the library to thermal denaturation and renaturation in the presence of a reducing agent before biopanning procedure. The scFv clones obtained after this treatment resulted the same isolated using standard biopanning conditions, suggesting that the selection efficiency of this repertoire is not affected by disulphide bonds formation. This evidence was confirmed by surface plasmon resonance analysis, measuring antigen affinity of a panel of library-derived scFv fragments both in oxidizing and reducing conditions. We observed perfectly comparable rate constants for antigen-scFv interactions in both antibody redox formats, demonstrating complete functionality also in the absence of intra-domain disulphide bonds. The experimental data point out that it is possible to straightforwardly isolate from this library scFvs with different specificities able to be functionally expressed in the cell cytoplasm. Hence, this library represents a valuable source of intrabodies for therapeutic applications.