Chronic Myeloid Leukemia (CML) is characterized by the BCR::ABL1 fusion gene, driving uncontrolled myeloid cell proliferation. Furthermore, metabolic dysregulation contributes to disease progression. Despite the efficacy of tyrosine kinase inhibitors (TKIs), unresolved clinical needs persist, necessitating refined preclinical models. This study compared responses of three commonly used CML cell lines (K562, LAMA84, KCL22) to five TKIs (imatinib, nilotinib, dasatinib, bosutinib, ponatinib) and a Specifically Targeting the ABL Myristoyl Pocket (STAMP) inhibitor commonly used in clinical settings. Using morphological assessments, viability and metabolic activity assays, glutamate intake evaluations, and gene expression analyses we observed distinct responses among cell lines. TKIs and STAMP inhibitor treatments showed varying impacts on morphological features, cell viability, metabolic activity, and gene expression profiles, highlighting significant differences in cellular responses. This emphasizes the necessity of considering cellular heterogeneity in CML research. This comprehensive comparison provides valuable insights for refining preclinical models and enhancing translational relevance in CML research and treatment development. Understanding the diverse responses of CML cell lines to TKIs and STAMP inhibitor facilitates the selection of appropriate models for specific research questions, ultimately improving the accuracy and reliability of preclinical studies in CML.