This study aimed to design and characterize mutant peptides targeting the CDK9/cyclin T1 protein-protein interaction, a crucial process in various pathological conditions such as HIV-1 infection and cancer. Using the Osprey software, 7,776 mutant peptides were generated based on the binding region of cyclin T1 to CDK9. After comprehensive analysis, three peptides (mp3, mp20, and mp29) were identified as promising inhibitors with high affinity and low free binding energy. These peptides exhibited favorable safety profiles and dynamic behaviors, interacting with a conserved sequence in CDK9 (residues 60–66). The peptides were designed to facilitate recombinant production in *Escherichia coli* using the plasmid vector pET28a (+). Predictions indicated good solubility upon overexpression, supporting their potential for downstream applications. The findings highlight the potential of these peptides as high-affinity CDK9 blockers, with further experimental studies needed to validate their biological activity and selectivity.This study aimed to design and characterize mutant peptides targeting the CDK9/cyclin T1 protein-protein interaction, a crucial process in various pathological conditions such as HIV-1 infection and cancer. Using the Osprey software, 7,776 mutant peptides were generated based on the binding region of cyclin T1 to CDK9. After comprehensive analysis, three peptides (mp3, mp20, and mp29) were identified as promising inhibitors with high affinity and low free binding energy. These peptides exhibited favorable safety profiles and dynamic behaviors, interacting with a conserved sequence in CDK9 (residues 60–66). The peptides were designed to facilitate recombinant production in *Escherichia coli* using the plasmid vector pET28a (+). Predictions indicated good solubility upon overexpression, supporting their potential for downstream applications. The findings highlight the potential of these peptides as high-affinity CDK9 blockers, with further experimental studies needed to validate their biological activity and selectivity.