Abstract <p>A new method for the simultaneous determination of Ni(II), Cu(II), and Co(II) ions in environmental samples by reversed-phase liquid chromatography was developed. The developed method is based on the synthesis of the quadrivalent Schiff base 6,6'-((1E,1'E)-(ethane-1,2-diylbis(azaneylylidene))bis(methaneylylidene))bis(3-methoxyphenol) by the condensation reaction between 2-hydroxy-4-methoxybenzaldehyde and ethylenediamine. The resulting Schiff base was used as a chelating agent to convert the targeted ions into metal-ligand complexes within 1 min, which were extracted on an octadecylsilane (<b>ODS</b>) solid-phase extraction mini-column and then injected into a reversed-phase high-performance liquid chromatography system. Sufficient separation of the metal-ligand complexes was achieved on a GL Sciences Intersil ODS-3 column with a mobile phase composition of methanol-acetonitrile-water (30 : 30 : 40, v/v) within 6 min and detection by an ultraviolet detector at 254 nm. The limits of detection were found to be 0.45, 0.40, and 1.47 µg/L for Co²⁺, Cu²⁺, and Ni²⁺, respectively. Recoveries from water samples ranged from 97.4 to 103.6%, with relative standard deviation values between 0.4 and 1.7%. While the targeted metal ions were not detected in a sample from the remote upstream area of the Kabaca Stream (Artvin, Turkey), concentrations were found to be 116.4 µg/L for Cu²⁺ and 32.9 µg/L for Ni²⁺ in a sample taken from the Kabaca Stream near the industrial area (Artvin, Turkey). Co²⁺ was below the limit of detection in both cases. In addition, the developed method was also validated using the inductively coupled plasma optical emission spectrometry technique.</p>