There are currently a limited number of techniques to study nickel speciation in aqueous samples. This work reports on the use of the permeation liquid membrane (PLM) for that objective. In this paper, the composition of the organic phase was studied to maximize the Ni flux (thus the sensitivity of the device) over a wide Ni2+ concentration range (50 nM to 100 μM) in order to verify its ability to determine free Ni2+ in the presence of Ni complexes. A mixture containing 1,10-didecyl-1,10-diaza-18-crown-6 ether (22DD) and di(2-ethylhexyl)phosphoric acid (D2EHPA) in toluene/phenylhexane was selected as the optimized organic phase for the PLM. The PLM was shown to be a reliable tool to measure free nickel concentrations down to 10−7 M. The effect of pH on Ni transport was also studied. Fluxes below pH 6 were reduced significantly, i.e. an order of magnitude smaller than fluxes above pH 7.8. Finally, as part of a broader study examining the ability of trace metals to induce antibiotic resistance in bacteria, we used the PLM to quantify the formation, at pH = 7.2, of a weak complex between Ni and Imipenem (a member of the carbapenem class of β-lactam antibiotics) or its hydrolysis product(s).