To investigate whether MELK phosphorylates Cofilin-1 at residue Threonine 70 (T70), we performed a phosphoproteomic validation using immunoprecipitation coupled with mass spectrometry (IP-LC-MS/MS). HEK293T cells were divided into three experimental groups: (1) MOCK control (Empty Vector), (2) MELK overexpression, and (3) MELK overexpression treated with the inhibitor OTSSP167. Endogenous Cofilin-1 was enriched by immunoprecipitation. The study aimed to identify and quantify the phosphorylation status of Cofilin-1, specifically focusing on the T70 site, and to assess the impact of MELK kinase activity and its inhibition by OTSSP167 on this phosphorylation event.