Leukemia is a malignant tumor of the hematological system characterized by the uncontrolled proliferation of abnormal hematopoietic cells in the bone marrow. It often presents with anemia， bleeding tendency， infection risk and organ invasion. These clinical symptoms bring severe survival risks to patients. Although traditional chemotherapy regimens are effective in the treatment of some hematological malignancies， their efficacy is limited for elderly patients， those with high-risk genetic characteristics or comorbidities. In recent years， targeted drugs have revolutionized the treatment of leukemia. By selectively inducing tumor cell apoptosis， they have significantly improved the remission rate and survival prognosis of patients with multiple leukemia subtypes. Venetoclax is a B-cell lymphoma 2 （BCL-2） inhibitor and plays an important role in the clinical treatment of hematological malignancies， such as acute myeloid leukemia and chronic lymphocytic leukemia. In addition， it also shows potential efficacy in other hematological malignancies such as multiple myeloma and mantle cell lymphoma. Although the efficacy of venetoclax is remarkable， the individual differences in blood drug concentration are significant due to factors such as drug interactions， polymorphisms of metabolic enzymes， and liver and kidney function. Venetoclax exhibits significant inter-individual pharmacokinetic differences， the trough concentration is significantly correlated with the treatment response， and if the peak concentration exceeds the warning concentration， adverse reactions will be triggered. Clinical trials have reported a variety of adverse events associated with venetoclax， including neutropenia， tumor lysis syndrome， thrombocytopenia， infection， anemia， diarrhea， nausea， upper respiratory tract infection， cough and musculoskeletal pain. Therefore， to minimize the risk of adverse events in the clinical use of venetoclax as much as possible， it is necessary to reasonably guide its clinical dosage. Therapeutic drug monitoring can optimize individual dosing regimens by measuring the steady-state concentration in patients’ blood. This research aims to establish a rapid， sensitive and reliable ultra performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS） method. This method is used for the quantitative determination of venetoclax in plasma， and its performance was validated. This method employs an electrospray ionization and multiple reaction monitoring （MRM） in the positive ion mode to detect venetoclax and its isotope internal standard venetoclax-d₈. Methanol was used for protein precipitation， C18 reversed-phase chromatographic column was used for liquid phase separation. Gradient elution was performed using acetonitrile and 0.1% formic acid aqueous solution as the mobile phases. The flow rate was 0.4 mL/min， the run time was 3.5 min， and the retention time of venetoclax was 1.95 min. The analysis time is short， facilitationg the rapid determination of clinical samples. Dosage escalation is commonly adopted in the treatment of venetoclax. In this study， a pretreatment approach involving extraction followed by dilution was used. This methodincreased the upper limit of quantification and expanded the linear range. The linear range of venetoclax was 50–10 000 ng/mL， r²>0.999. The method had good specificity. The intra-run precision and inter-run precision were 1.8%–4.5% and 2.7%–6.1%， respectively， the recoveries were 100.3%–102.9%， the matrix effects ranged from 88.0% to 111.0%， and the carryover was less than 20% of the minimum concentration of linear range. This method was applied to the therapeutic drug monitoring of venetoclax in acute myeloid leukemia （AML） patients， and the peak and trough concentrations of venetoclax were obtained for different patients to monitor their blood drug concentration. The above research results indicate that this method can accurately and robustly quantify venetoclax in human plasma， which helps address the drug monitoring needs of leukemia patients receiving venetoclax treatment and guide clinical personalizd therapy.