AbstractKinetochores are multi‐protein assemblies present at the centromere of the human chromosome and play a crucial role in cellular mitosis. The CENP‐T and CENP‐W chains form a heterodimer, which is an integral part of the inner kinetochore, interacting with the linker DNA on one side and the outer kinetochore on the other. Additionally, the CENP‐T‐W dimer interacts with other regulatory proteins involved in forming inner kinetochores. The specific roles of different amino acids in the CENP‐W at the protein‐protein interaction (PPI) interface during the CENP‐T‐W dimer formation remain incompletely understood. Since cell division goes awry in diseases like cancer, this CENP‐T‐W partnership is a potential target for new drugs that could restore healthy cell division. We employed molecular docking, binding free energy calculations, and molecular dynamics (MD) simulations to investigate the disruptive effects of amino acids substitutions in the CENP‐W chain on CENP‐T‐W dimer formation. By conducting a molecular docking study and analysing hydrogen bonding interactions, we identified key residues in CENP‐W (ASN‐46, ARG‐53, LEU‐83, SER‐86, ARG‐87, and GLY‐88) for further investigation. Through site‐directed mutagenesis and subsequent binding free energy calculations, we refined the selection of mutant. We chose four mutants (N46K, R53K, L83K, and R87E) of CENP‐W to assess their comparative potential in forming CENP‐T‐W dimer. Our analysis from 250 ns long revealed that the substitution of LEU83 and ARG53 residues in CENP‐W with the LYS significantly disrupts the formation of CENP‐T‐W dimer. In conclusion, LEU83 and ARG53 play a critical role in CENP‐T and CENP‐W dimerization which is ultimately required for cellular mitosis. Our findings not only deepen our understanding of cell division but also hint at exciting drug‐target possibilities.