The aim of the study was application of a high performance liquid chromatography (HPLC) with UV detector for selenium speciation in samples of mineral water. Basic solutions 1000μg/l SeO₃^2- and SeO₄^2- were prepared by dissolving 2,190 g of Na₂SeO₃ and 2,393 g Na₂SeO₄ in 1000 ml of water, respectively. Recent standards SeO₃^2- and SeO₄^2- (single or mixed) were prepared every day from the basic separation of Se(IV) and Se(VI) was obtained using a C18 column (250×4.0mm; 5μm) with mobile phase comprising 5mmol/lphosphate tetrabutylammonium, 50/50 water.methanol at pH 3,4. Flow rate was 1ml/min. Detector was set at 210 nm. Injection volume was 5 μl. Retention time of selenium(IV) was 2.27 min and of selenium(VI) 4.37 min. Recovery of Se determined using a HPLC method was 99.6 %. The determined content of Se(IV) was 4,10 μg/l and Se(VI) – 4,27 μg/l. The method worked out can be therefore recommended for determination of Se in samples of mineral water.