Traditional drug therapies often target a limited number of conserved sites, such as ATP-binding regions. However, the use of these sites often leads to off-target toxicity in both malignant and normal cells, limiting therapeutic specificity. Current research on non-ATP inhibitors can identify more effective drug targets that promote selective apoptosis, specifically in cancer cells. This study is using yeast (Saccharomyces cerevisiae) as a model system to investigate protein interactions and design specific peptides with the intention to disrupt interactions specific to cancer cells. The objective of this project is to detect interactions between retinoblastoma (RB1) and human Cyclin A (CCNA2) in Yeast-2-Hybrid (Y2H) and Yeast-3-Hybrid (Y3H) systems. CCNA2 and RB1 were cloned into two different expression vectors, pGBK and pGAD. Once the CCNA2-RB1 interaction is detected using the activation of reporter genes in a Y2H system, a third plasmid that encodes for the amino acid sequence HAKRRLIF will be introduced. This peptide sequence was previously demonstrated to disrupt the CCNA2-RB1 interaction in vitro, so the expectation is that it will disrupt the protein-protein interaction in the Y3H system as well. Following this proof of principle, an oligo/peptide library will be generated to develop a high-throughput screen for disruption of any combination of protein interactions. Promising candidates will be converted into peptide-mimetic compounds. Then, the inhibitory drug-like compounds will be optimized into pharmaceutically relevant compounds using REPLACE (Replacement with Partial Ligand Alternatives through Computational Enrichment) technology. Using the Y3H system, drug-like compounds can be developed with the ability to disrupt protein interactions specific to cancer cells.