BACKGROUND Therapeutic drug monitoring is recommended for the optimal management of patients with several malignant diseases. The aim of this study was to develop and validate an isotope dilution direct injection mass spectrometry method for the high throughput determination of tyrosine kinase inhibitors in plasma from leukemic and cancer patients. METHODS The plasma for analysis was deproteinated by methanol and the centrifuged supernatant was directly injected to mass spectrometer without separation step. Multiple reaction monitoring modes on a hybrid triple quadrupole - linear ion trap mass spectrometer (5500 QTRAP) were used for the detection and quantification of imatinib, nilotinib, lapatinib, and dasatinib. RESULTS We developed a fast method with analysis time of 55 s and 19s in multiple injection setting. The method was successfully validated and applied to the patient plasma samples. In order to overcome insufficient sensitivity of dasatinib, multiple reaction monitoring cube mode in linear ion trap (MRM(3)) was successfully applied. The limits of quantification were in the range 1.0-5.5 ng/ml. Imprecisions were lower than 6.9% and the accuracy of the quality control samples ranged between 99.0 and 107.9%. CONCLUSIONS Isotope dilution direct injection mass spectrometry method allows high-throughput therapeutic drug monitoring of tyrosine kinase inhibitors in plasma. The method offers low-cost analyses as a result of its speed and the exclusion of separation step and can be advantageously used in routine clinical practice. The method can be applied on various drugs and biochemical markers with the use of triple quadrupole instruments.