Valinomycin is an antibiotic which is a cyclic macro molecule made up of twelve alternating amino acids, D-Valine, L-Valine, D-Hydroxyisovaleric acid and L-Lactic acid. It is a transport protein that selectively binds to potassium ions and transports them across the cell membrane. We have used different ONIOM methods to optimize Li⁺, Na⁺ and K⁺-valinomycin complexes, where the central parts of the complex involved in the interaction between the metal ion and valinomycin were optimized with higher level theories. The computational calculations are supported by experimental analysis using a triple quadruple molecular mass analyzer with an atmospheric pressure electrospray ionizer source. We sampled the alkali ion-valinomycin complexes in solution to investigate equilibrium constants. The selectivity of valinomycin for Li⁺, Na⁺, K⁺, Rb⁺, and Cs⁺ is a function of the solvent environment. In the gas phase valinomycin binds selectivity to Li⁺>Na⁺>K⁺ and in solution valinomycin competes with solvent to bind with the alkali ion.