A simple, accurate and reproducible reversed-phase high performance liquid chromatography method with fluorescence detection was developed for determination of octylphenol, nonylphenol and their short-chain ethoxylates, using an octadecyl silica column. The mobile phase used for the analysis consisted of methanol, acetonitrile and water (50:15:35). The analytes were separated successfully within a reasonable time without gradient elution, which was not achieved previously. Thus, use of inexpensive fluorescence detection instead of MS detection was enabled. This chromatographic method, combined with solid-phase extraction, was highly applicable for the simultaneous determination of octyl- and nonylphenol as well as their mono- and diethoxylates in real river water samples. The limit of detection (LOD) for octylphenol and its ethoxylates was 0.05 μg · L −1 . For nonylphenol the LOD was 0.14 μg · L −1 and for its ethoxylates it was 0.15 μg · L −1 . Recovery of octylphenol and its ethoxylates was about 80%. Recovery of nonylphenol and its ethoxylates was slightly lower (from 60 to 76%).