Monopolar Spindle 1 (MPS-1) and Aurora B (AurB) are highly conserved mitotic kinases that are required for proper chromosome alignment and accurate chromosome segregation. Previous investigations identified similar defects in chromosome alignment after inhibition of either AurB or MPS-1, thus suggesting that the two kinases might be redundant. Based on their redundant roles in chromosome alignment and motility, it has been proposed that MPS-1 and AurB kinase activities might regulate the same substrate. To test that hypothesis MPS-1 inhibition was achieved using reversine and compared to AurB inhibition by ZM447439. Inhibition of MPS-1 perturbed recruitment of the fibrous corona, affecting dynein, dynactin, the ROD-ZW10-Zwilch (RZZ) complex, spindly,   BubR1, Mad1 and Mad2 but not Zwint-1 or HEC-1 kinetochore localization. This defined the boundary of MPS-1 activity in between Zwint and the RZZ complex, similar to that seen with AurB inhibition. In vitro kinase assays were used to identify candidate substrates and identified Zwilch, but not Zw10 or Zwint, as a novel substrate for MPS-1. LC/MS/MS analysis of phosphorylated Zwilch revealed 3 novel phosphorylation sites which were further validated by in vitro kinase assay. Expression of a triple-Ala (3A) mutant of Zwilch induced defects similar to MPS-1 inhibition and led to a loss of dynein, dynactin, spindly, RZZ complex and anaphase inhibitors from the kinetochores. A triple-Glu (3E) mutant of Zwilch rescued the effects of MPS-1 inhibition by reversine. Recombinant GST-Zwilch-3E mutant was able to pull down Zw10 from mitotic cell lysates unlike wild type GST-Zwilch. These studies suggest that MPS-1 and AurB are non-redundant kinases, where MPS-1 phosphorylation of Zwilch is required for proper assembly of the fibrous corona during prometaphase through its interaction with Zw10, placing Zwilch and RZZ at the center of checkpoint signaling and chromosome alignment.