BCR-ABL1-based resistance to imatinib, mainly resulting from BCR-ABL1 mutations, is largely solved after second- and third-generation tyrosine kinase inhibitors (TKIs) are discovered. Nonetheless, imatinib resistance without BCR-ABL1 mutations, including intrinsic resistance induced by stem cells within chronic myeloid leukemia (CML), remains the major clinical challenge for many patients.To study the key active ingredients and corresponding target proteins in Huang-Lian-Jie-Du-Tang (HLJDT) against BCR-ABL1-independent CML resistance to therapeutics, and then explore its mechanism of against CML drug resistance.Cytotoxicity of HLJDT and its active ingredients in BCR-ABL1-independent imatinib resistance cells was analyzed through MTT assay. The cloning ability was measured through soft agar assay. Monitoring therapeutic effect on Xenografted mice CML model by in vivo imaging technology and mice survival time. Predicting the potential target protein binding sites by the technology of photocrosslinking sensor chip, molecular space simulation docking, and use Surface Plasmon Resonance (SPR) technology . Flow cytometry to detect the ratio of stem progenitor cells (CD34+). Constructing bone marrow transplantation mice CML leukemia model, detect the effects on leukemia stem cells LSK (Lin-\ Sca-1+ \C-kit+) self-renewal.Treatment with HLJDT, berberine and baicalein inhibited cell viability and colony formation of BCR-ABL1-independent imatinib-resistant cells in vitro while prolonging survival in mouse with CML xenografts and transplatation CML-like mouse models in vivo. JAK2 and MCL1were identified as targets of berberine and baicalein. JAK2 and MCL1 are involved in multi-leukemia stem cell-related pathways. Moreover, the ratio of CD34+ cells in resistant CML cells is higher than in treatment-sensitive CML cells. Treatment with BBR or baicalein partially suppressed CML leukemic stem cells (LSCs) self-renewal in vitro and in vivo.From the above, we concluded that HLJDT and its key active ingredients (BBR and baicalein) allowed to overcome imatinib resistance with BCR-ABL1 independent by eradication of LSCs by targeting the JAK2 and MCL1 protein levels. Our results lay the foundation for applying HLJDT in patients with TKI-resistant CML.Copyright © 2023 Elsevier GmbH. All rights reserved.