Through screen and subsequent validation, we reveal substrates of RSK2 and PAK4 kinases as main effectors in responding to 5‐FU chemotherapy, and combinational treatment of colon cancer cells with 5‐FU and RSK2 inhibitor or PAK4 inhibitor can largely inhibit cell growth and enhance cell apoptosis. Here, RPS6KA3 is linked to malignant colon neoplasm.