Olanzapine, second-generation antipsychotic, has high clinical potential in removing symptoms of schizophrenia. Because of very low extrapyramidal toxicity, olanzapine is drug of choice, instead of conventional antipsychotic drugs. On the other hand, because of possible side effects at therapeutic doses, interindividual variability of pharmacokinetics, poor patient adherence and potential drug-drug interactions, careful dose titration is necessary for every patient. Individualization of therapy is based on periodical following of blood drug concentration with sensitive and selective methods such as high performance liquid chromatography. Aim of this study was to examine reliability of HPLC method in quantification of olanzapine. Validation of parameters for specific quantitative in-house methods was performed, namely: selectivity, linearity, limit of detection and quantification (LOD and LOQ), accuracy and precision (repeatability and intermediate precision). For method validation commercial samples and serum samples of patients from departments of psychiatry were used. Based on measurement and data analysis it was concluded that the method provides linear response in the range of 4,8 – 960 nmol/L, which includes therapeutic reference range for olanzapine in blood (64-256 nmol/L). Selectivity of the method is confirmed by a diode array detector based on comparing of UV-spectra of olanzapine. A LOQ value of 7,796 nmol/L and LOD of 2,732 nmol/L were also defined. The precision and accuracy results met the set criteria. However, day-to-day precision was examined only during 12 days. Additionally, verification of therapeutic reference range for olanzapine in blood is conducted on 58 patients' samples. Concentration of olanzapine in blood does not follow normal distribution and values are shifted to lower concetrations of reference range. Status of smoking, additional therapy and other intra- and interindividual variabilities were not considered so these results were expected. Method has shown satisfactory validation and clinical application, but it is desirable to examine intermediate precision during longer period of time. Also, it should be kept in mind that this validation did not include evaluation of stability of samples. Accuracy of measurements and reliability of validation results are additionally confirmed by regular participation of laboratory in external quality control assessment.