Objectives: Development of a method for the simultaneous determination of carbamazepine, lamotrigine and phenobarbital by using highly effective liquid chromatography, to determine the optimal conditions for the determination of antiepileptics by high performance liquid chromatography and to investigate the efficacy of the chromatographic method on biological samples of blood. Design: Experimental study Settings: Laboratory of toxicology, Department of pathology, medicine and cytology, University Hospital of Split Materials and methods: Lamotrigine, phenobarbital and carbamazepine solutions were prepared and the appropriate mobile phase was mixed, by mixing the buffer, methanol and acetonitrile in various ratios. The parameters for selecting the optimal method for simultaneous determination of all three antiepileptics using the HPLC method were investigated, including a change of wavelength (306 nm, 270 nm and 220 nm), temperature (30 ° C and 40 ° C) and mobile phase flow (0.5 - 1.4 mL / min). Biological blood samples, supplemented with drug solutions, were extracted by liquid-liquid (LLE) method and injected into an HPLC device. Results: HPLC analysis has successfully demonstrated the presence of lamotrigine on all three wavelengths investigated, with the most pronounced response at wavelength of 270 nm, while the presence of phenobarbital is only proved when measuring at wavelength 270 nm. For that reason, the wavelength of 270 nm was selected for further testing of carbamazepine, whose presence under these conditions has been successfully proved. Adapting the mobile phase flow shortened the dead time of the analysis. By applying the developed method to real biological blood samples simultaneously, all three antiepileptics tested were determined . Conclusion: High performance liquid chromatography was successfully used for simultaneous determintaion of three antiepileptics: lamotrigine, phenobarbital and carbamazepine. An optimal method and parameters for qualitative and quantitative analysis of three antiepileptics were determined separately and in biological samples. It is of great importance to develop methods for determining the concentration of antiepileptics mainly due to adaptation of patient therapy. For further research it is recommended to use a greater number of antiepileptics to determine the most efficient and quickest method applicable to daily laboratory work.