Ribosome biogenesis is a hallmark of cancer and has emerged as an effective target for treatment. This study investigates novel strategies in cancer therapy by disrupting specific protein-protein interactions essential for the ribosome biogenesis process. Using peptide mimetics derived from human Bop1, conjugated with cell-penetrating sequences, we assessed their binding affinity, cellular internalization, and biological effects in HCT116 colon cancer cells. The peptides disrupted Bop1-WDR12 interaction, leading to nucleolar stress, reduced ribosome biogenesis and decreased protein synthesis. Importantly, these interventions triggered apoptosis via caspase activation without eliciting DNA double-strand breaks. These findings demonstrate that targeted disruption of ribosome assembly pathways can induce cancer cell death independently of genotoxicity, offering a promising non-genotoxic therapeutic approach that would minimize side effects. Targeting protein-protein interactions of assembly factors during ribosome maturation can be a new strategy with significant potential to improve cancer treatments by selectively impairing tumor growth while not contributing to genomic instability.