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Topiramate (TPM) is a new antiepiletic drug with efficacy in several types of seizures. Therapeutic drug monitoring of TPM is essential for effective patient management. The aim of this study was to evaluate the use of dried blood spot (DBS) specimens to determinate the TPM levels during the treatment. Advantages of DBS include short collection time, low invasiveness, ease and low cost of sample collection, transport and storage. Performance comparison between this method and the commercially available fluorescence-polarization immunoassay (FPIA) was made.The analysis was performed in selected reaction monitoring (SRM) mode. The calibration curve in matrix using D12-topiramate was linear in the concentration range of 0.0166–1.66 mg/L (0.5–50 mg/L in DBS) of topiramate with correlation coefficient value of 0.9985. In the concentration range of 0.5–50 mg/L, the coefficients of variation in DBS were in the range 2.13–11.85% and the accuracy ranged from 93.93% to 110.67%.There was no significant differences between the concentrations (ranging 0.5–50 mg/L) measured both FPIA on venous samples and LC-MS/MS assay on simultaneous DBS samples.The sensitivity and specificity of tandem mass spectrometry allow now high throughput topiramate analysis (the improvement was three times in comparison with FPIA). This new assay has favourable characteristics being highly precise and accurate. FPIA also proved to be precise and accurate, but is not always suitable for the sample collection in neonates in whom obtaining larger blood samples is not convenient or possible.