Introduction: Imatinib has favorable pharmacokinetic properties, but primary and secondary resistance mechanisms may cause a decrease in clinical response over time. There is a positive correlation between serum imatinib concentrations and treatment response. Our aim was to develop a method for the measurement of imatinib and its' active metabolite N-desmethyl imatinib. Methods: Serum imatinib and N-desmethyl imatinib levels were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and validation studies were carried out according to CLSI (The Clinical & Laboratory Standards Institute) protocols. Serum samples were collected from 40 patients with chronic myeloid leukemia (CML) and analyzed with LC-MS/MS and ultra high-performance liquid chromatography (UHPLC) methods. Results: The linearity range and correlation coefficient were 12.2–12,500 ng/mL and 0.9987 for LC-MS/MS method, respectively. Limit of quantitation was determined as 24.4 ng/mL. The retention times of imatinib and N-desmethyl imatinib were 1.66 and 1.60 min, respectively. There was no statistically significant difference between the results of both methods. Discussion: This LC-MS/MS method is cost-effective and has adavantages such as using low serum volumes, requiring simple pretreatment steps (only protein precipitation) and reduced turnaround times for analysis.