Imatinib is an oral tyrosine kinase inhibitor and first-line therapy for chronic myeloid leukemia (CML) patients. There is a positive correlation between serum imatinib concentrations and treatment response. However, the specific relationship between plasma concentration of imatinib and its influencing factors remains unclear. This study collected the basic information of 102 patients using imatinib as the first-line CML treatment drug. Further, we analyzed the individual differences in imatinib concentration and explored its influencing factors. Through the intra-day and inter-day precision studies, we found the precision for imatinib assay methodology was both within ± 13%, and the recovery rate was above 85%. The blood concentration of imatinib had a noticeable individual difference, and the recommended treatment concentration is 860-1500 ng/mL, with only 41.40% of patients achieving this concentration. Meanwhile, there was a negative correlation between age and imatinib trough concentration (Ctrough), the same as that between age and N-desmethyl imatinib. Besides, compared with the teenage group, the serum imatinib Ctrough aged 17∼47 years and 48∼68 years were significantly reduced. Further analysis shows that imatinib Ctrough values reached therapeutic concentrations (59%) increased dramatically in CML patients from 17 to 47 years. Moreover, the dose groups of 400 mg/d resulted in therapeutic imatinib concentrations in 68% of CML patients, which was the best. The established method was validated with acceptable accuracy, precision, linearity, and stability as required, which was successfully applied to the TDM of imatinib. Age, dose, and metabolites can influence the imatinib concentration and its therapeutic effect in CML patients. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.