INTRODUCTION AND OBJECTIVES: Colorectal cancer (CRC) is the second most common type of cancer in Brazil. The 5-year survival rate of advanced-stage patients is only 11-12%. One of the main characteristics of tumors is the dysregulation of the cell cycle. New therapies, which target cell cycle enzymes, have emerged in the last decade. Among such molecular targets are the cyclin-dependent kinase (CDK) 4/6 inhibitors, Palbociclib and Abemaciclib. These inhibitors have been used to treat advanced breast cancer that expresses Retinoblastoma (RB), the primary target of the CDK 4/6. CRC has changes in the RB pathway very similar to breast cancer: amplification of CCNE1, CDK4, and CDK6, such as loss of CDKN2A Therefore, it is a potential candidate for CDK4/6 inhibitor therapy. This work aims to evaluate the efficacy of new CDK4/6i agents as monotherapy or in combination with first-line chemotherapy in CRC. The use of these new inhibitors can optimize treatment regimens regarding how to combine these drugs with others and what resistances may arise. MATERIAL AND METHODS: CRC (SW480 and HCT116) cell lines were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum and incubated at 37ºC and 5% CO2. Dose-response curve analysis of first-line chemotherapy drugs in CRC, SN-38 (the active metabolite of irinotecan), oxaliplatin, and 5-fluorouracil (5-FU), was performed to determine low doses that resulted in 80% of cell viability (EC20) to be combined with effective CDK4/6i doses in the high-content screening platform using the Live/Dead kit. RESULTS AND CONCLUSION: The EC20 was determined in the colorectal lines HCT116 and SW480 after 48 hours of treatment with first-line chemotherapy for further combinations with CDK4/6i. In preliminary results, HCT116 cells treated with the combination of abemaciclib (726 nM) or palbociclib (360 nM) with 0.2µM of oxaliplatin showed a decrease in viable cells compared to either drug alone. The combinations of abemaciclib (726 nM) with 3.4 µM of 5-Fu or 1.1 nM of SN-38 are also effective in reducing the percentage of living cells compared to each drug alone. We are currently testing the combination of CDK4/6i with chemotherapy for other cell lines. These results suggest that abemaciclib might be beneficial to treat colorectal cancers that express RB.