Chemical fractionation and speciation of metals species in natural waters and its relation with bioavailability have received increased attention in recent years. A simple liquid membranes method, based on coupled liquid extraction and re-extraction processes, is proposed to separate and quantify the species of nickel present in water samples. A simplex optimization of chemical variables, such as carrier concentration in the organic solution and nitric acid concentration in the receiving solution, was performed and, under optimized conditions, the extraction system was applied to determine nickel species in water samples at natural level concentrations. A linear relationship was established between extraction efficacy and the concentration of dissolved organic carbon in the samples, allowing the separation and determination of labile and non-labile nickel fractions, since the latter was not transported through the organic solution acting as liquid membrane. When the total and labile concentrations of metals were analyzed in real samples with different salinities, no significant differences were found between the results obtained and those from well-established methods. An average relative error of 1.50 and 2.37 was obtained for total Ni concentration and labile fraction, respectively. Finally, a comparison with the theoretical speciation data calculated with the software WinHumic V was successfully performed. Thus, the proposed method allows the simultaneous determination of labile and non-labile nickel fractions, presented as a simple alternative to nickel fractionation in natural waters.