Progression through mitosis is balanced by the timely regulation of phosphorylation and dephosphorylation events ensuring the correct segregation of chromosomes before cytokinesis. This balance is regulated by the opposing actions of CDK1 and PP2A, as well as the Greatwall kinase/MASTL. MASTL is commonly overexpressed in cancer, which makes it a potential therapeutic anticancer target. Loss of Mastl induces multiple chromosomal errors that lead to the accumulation of micronuclei and multilobulated cells in mitosis. Our analyses revealed that loss of Mastl leads to chromosome breaks and abnormalities impairing correct segregation. Phospho-proteomic data for Mastl knockout cells revealed alterations in proteins implicated in multiple processes during mitosis including double-strand DNA damage repair. In silico prediction of the kinases with affected activity unveiled NEK2 to be regulated in the absence of Mastl. We uncovered that, RAD51AP1, involved in regulation of homologous recombination, is phosphorylated by NEK2 and CDK1 but also efficiently dephosphorylated by PP2A/B55. Our results suggest that MastlKO disturbs the equilibrium of the mitotic phosphoproteome that leads to the disruption of DNA damage repair and triggers an accumulation of chromosome breaks even in noncancerous cells.