Abstract Background and Purpose: Cbl-b, a RING-type E3 ubiquitin ligase in the Casitas B-lineage lymphoma protein family, negatively regulates immune responses. Its inhibition enhances immune cell function, aiding in combating pathogens and tumors. This study aimed to discover novel small-molecule Cbl-b inhibitors and evaluate their potential in modulating immune activity. Experimental approach: A high-throughput screening of a self-constructed compound library identified hit compounds. Their interaction with Cbl-b was validated at the molecular level, and molecular docking confirmed binding sites. In vitro phosphorylation and ubiquitination systems were used to explore the inhibitory mechanism of 450F10, with cellular activity assessed in Jurkat cells and CD8+ T cells. Key Results: It was demonstrated that 450F10 binds specifically to F263 and Y260 of Cbl-b, thus inhibiting the phosphorylation-dependent activation of Cbl-b, thereby suppressing its protein activity. Notably, It enhances interleukin-2 (IL-2) secretion by human peripheral CD8+ T cells, boosting immune responses. Conclusion and implications: This study identified 450F10 as a novel small-molecule Cbl-b inhibitor capable of enhancing TCR signal stimulation and promoting IL-2 secretion in CD8+ T cells without affecting cell proliferation at effective concentrations. 450F10 shows potential as a valuable tool for exploring Cbl-b’s role in immune regulation. Future efforts will focus on optimizing the compound’s structure to enhance its activity and evaluating its efficacy and safety in animal models. K E Y W O R D S: Cbl-b, High-Throughput Screening, T Cell Activation, Small Molecule Inhibitors, Immunotherapy