Recent experimental studies have demonstrated that incorporating polyproline segments into arginine (R)‑rich cell‑penetrating peptides (CPPs) enhances their membrane penetration efficiency. To investigate this effect, we designed a polyproline-containing R-rich peptide, P9R9, and examined the contribution of proline residues to membrane translocation through in silico design and molecular dynamics (MD) simulations. Using the weighted ensemble method within the MD framework, we elucidated how the polyproline segment modulates the spontaneous translocation behavior of R-rich peptides. Notably, the penetration efficiencies predicted by in silico modeling did not consistently align with those observed in MD simulations, highlighting the mechanistic insights provided by dynamic simulations beyond those obtainable from static models.