Transcription Block Survival (TBS) is a high-throughput, intracellular peptide screening platform to derive functional transcription factor antagonists. TBS uses an entirely tag-free genotype-to-phenotype approach, selecting for desirable attributes such as high solubility, target specificity and low toxicity within a complex cellular environment. The assay has been validated against a number of transcriptional regulators including cJun, which is overactive in a range of diseases. Intracellular library screening against cJun identified a sequence that both binds cJun and antagonises function, which has been optimised through rational design. We have subsequently expanded the TBS methodology by screening libraries in a non-reducing E. coli cell line to facilitate covalent inhibition. A ~130,000 member peptide library was generated which included cys residues at 9 positions in the sequence. TBS screening selected for a peptide which formed a disulphide with a target cys within the cJun DNA-binding domain. In vitro biophysical characterisation confirmed the formation of the antagonist-target disulphide and its efficacy and the peptide has been further optimised by substitution of cys for covalent warheads towards the formation of an irreversible cJun antagonist.