Polo-like kinase 1 (Plk1) is a serine/threonine kinase involved in regulating the cell cycle. It is activated by aurora kinase B along with the cofactors Borealin, INCE, and survivn. Plk1 is involved in the development of resistances to chemotherapeutics such as doxorubicin, taxol, and gemcitabine. It has been shown that patients with higher levels of Plk1 have lower survival rates. Onvansertib is a competitive ATP inhibitor for Plk1 in clinical trials for the treatment of tumors and has recently entered a trial for the treatment of KRAS mutant colorectal cancers (CRCs). In this study, we have conducted an untargeted liquid chromatography-mass spectrometry (LC-MS) proteomics study as well as an untargeted lipidomics analysis of HCT 116 spheroids treated with onvansertib over a 72-hour time course experiment. Mass spectrometry Imaging (MSI) showed that onvansertib begins to accumulate after 12-hours, followed by decreasing and clearing out of the spheroid after 72-hours. Proteomics results showed alterations to cell cycle control proteins and an increasing abundance of aurora kinase B and Borealin. The proteomics data also showed alterations to many lipid metabolism enzymes. The MSI lipidomics data showed alterations to PC lipids, with many lipids increasing in abundance over time or increasing until 12-hours of onvansertib treatment and decreasing after that time point. In summary, these results suggest that onvansertib is causing the cells within the spheroid to become stuck in a certain phase of the cell cycle, quite possibly the S phase with the alterations in cell cycle control proteins and PC lipid alterations.