This study presents the design and characterization of three peptide inhibitors targeting the CDK9/cyclin T1 protein-protein interaction. The researchers used structure-based approaches to generate a library of 7,776 mutant peptides based on the binding region of cyclin T1 to CDK9. Through computational analysis, three peptides—mp3, mp20, and mp29—were identified as promising inhibitors with high affinity for CDK9 and low free binding energy. These peptides exhibited favorable safety profiles and demonstrated the ability to penetrate cells, suggesting their potential for intracellular activities. The study also revealed that mp3 and mp29 interact with a conserved sequence in CDK9 (residues 60–66). The peptides were cloned into the E. coli pET28a (+) vector for future recombinant production. Predictions indicated good solubility upon overexpression, supporting their potential for downstream applications. While the results demonstrate the promise of the designed peptides as CDK9 blockers, further experimental studies are needed to validate their biological activity and assess their selectivity. The findings highlight the potential of the designed peptides as high-affinity CDK9 inhibitors and contribute to the development of novel strategies for CDK9 inhibition.This study presents the design and characterization of three peptide inhibitors targeting the CDK9/cyclin T1 protein-protein interaction. The researchers used structure-based approaches to generate a library of 7,776 mutant peptides based on the binding region of cyclin T1 to CDK9. Through computational analysis, three peptides—mp3, mp20, and mp29—were identified as promising inhibitors with high affinity for CDK9 and low free binding energy. These peptides exhibited favorable safety profiles and demonstrated the ability to penetrate cells, suggesting their potential for intracellular activities. The study also revealed that mp3 and mp29 interact with a conserved sequence in CDK9 (residues 60–66). The peptides were cloned into the E. coli pET28a (+) vector for future recombinant production. Predictions indicated good solubility upon overexpression, supporting their potential for downstream applications. While the results demonstrate the promise of the designed peptides as CDK9 blockers, further experimental studies are needed to validate their biological activity and assess their selectivity. The findings highlight the potential of the designed peptides as high-affinity CDK9 inhibitors and contribute to the development of novel strategies for CDK9 inhibition.