Background: Multiple activating genetic mutations in the phosphatidylinositol-3 kinase (PI3K) and the mitogen activated protein kinase (MAPK) pathways have been implicated in the development of resistance to anti-cancer therapies. Ribociclib has limited activity as a single agent in CRC. However, combining Ribociclib with targeted therapies of the MAPK and PI3K pathways may be a promising treatment strategy for CRC. Methods: We explored the in vitro efficacy of drug combinations Ribociclib (R) and Alpelisib (A) in four CRC cell lines with different mutational status; CACO2 (PIK3CA/KRAS wild-type), LS1034 (KRAS mutated), SNUC4 (PIK3CA mutated) and DLD1 (PIK3CA/KRAS mutated). Drug combination index (CI) was calculated using Calcusyn Biosoft software. We used a western immunoblotting method for protein analysis. The Chick Chorio-Allantoic Membrane (CAM) assay was used for in-vivo analysis of the effect of the drug combination. Results: IC₅₀s for R and A were calculated for all four cell lines. CACO2 and DLD1 cells were resistant to Ribociclib (IC₅₀> 15µM). The cell lines had varying sensitivity to both drugs. Drug combination analysis showed that the combination of R and A has a synergistic anti-proliferative effect in all CRC cell lines tested. The combination of R and A is highly synergistic in LS1034 cells which harbour a KRAS mutation (CI = 0.16). Relative expression of the proteins Cyclin D1, E2F-1, p-BCL-2, p-AKT, p-Rb and p-S6 was determined by measuring the density of each band from the western blot experiments and normalizing to β-actin. Combined inhibition of CDK4/6 and PI3Kα caused a simultaneous reduction of p-RB, p-AKT and p-S6 and a more complete inhibition of the PI3K/AKT/mTOR pathway in all four cell lines tested. There was also an increase in the expression of the apoptotic marker pBCL2 in cells treated with the combination of R and A compared to vehicle control(VC). We observed a macroscopic reduction of visible tumour in all cell lines treated with the R and A combination in vivo as compared to VC. Microscopic examination also showed less Ki67 staining and negative cytokeratin staining in combination treated groups. Conclusions: We see a synergistic response to treatment with the combination of R and A in all cell lines and this combination may be a rational treatment strategy in colorectal cancer.