Background/Aim. Cyanobacterial toxins befall a group of various compounds according to chemical structure and health effects on people and animals. The most significant in this large group of compounds are microcystins. Their presence in water used for human consumption causes serious health problems, liver beeing the target organ. Microcystins are spread all over the world. Waterblooms of cyanobacterias and their cyanotoxins are also common in the majority of surface waters in Serbia. The aim of this study was to propose HPLC method for determination of mikrocystin-LR, to validate the method and to use it for determination of microcystin-LR in the surface water of the river Ponjavica. The Ponjavica is very eutrophic water and has ideal conditions for the cyanobacterial growth. Methods. Sample of water form the Ponjavica river were collected during the summer 2008. Coupled columns (HLB, Sep-Pak), were used for sample preparation and HPLC/PDA method was used for quantification of microcystin- LR. Results. Parameters of validation show that the proposed method is simple, fast, sensitive (0.1 mg/L) and selective with the yield of 89%-92%. The measuring uncertainty of 􀁲 5% was obtained. The obtained results for surface water show that microcystin concentration reached the maximum level during August and September (1.5 μg/L). The value is higher than maximum allowable concentration of microcystin in drinking water (1 μg/L) proposed by WHO. Conclusion. This study contributes to the issue of pollution of the National Park Ponjavica. Besides, literature data and WHO clearly point out harmfulness of cyanobasterias and their toxins and implicate the necessity of legislation concerning determination and monitoring of these toxins in our country. Method used for quentification of mycrocystin-LR was shown to be sensitive, selective, rapid and simple and could be recommended for routine determination of this toxin. [Projekat Ministarstva nauke Republike Srbije, br. III46009]