The microtubule is regarded as the key target for designing anticancer and neurotherapeutic drugs due to its functional importance in eukaryotic cells including neurons. The microtubule is a dynamic hollow polymer tube consisting of alpha,beta-tubulin heterodimer. Polymerization of alpha,beta-tubulin heterodimer resulted in microtubule formation. GTP plays a crucial role in microtubule polymerization. It binds at the exchangeable binding site of the beta-tubulin heterodimer, and it is one of the most crucial therapeutic hot spots for designing anticancer therapeutics. In this manuscript, we have shown using an in silico strategy and various in vitro and cellular experiments that the binding affinity to the tubulin and cancer therapeutic potential of an exchangeable GTP/GDP binding antimitotic tetrapeptide (SP: Ser-Leu-Arg-Pro) is increased through changing proline with the multifluorine substituted proline. This study showcases the importance of the proline amino acid and its pyrrolidine ring in the regulation of binding with tubulin at the GTP binding pocket.