The ability of alternative aqueous biphasic systems (ABS) composed of polyethylene glycol and imidazolium-based ionic liquids (ILs) to selectively separate similar biomolecules was here investigated. The preferential partitioning of three alkaloids (caffeine, xanthine and nicotine) was addressed by means of their partition coefficients and selectivity values. Aiming at optimizing the selectivity of the studied ABS, factors such as the chemical structure of the IL (cation side alkyl chain length, number of aliphatic moieties or their functionalization, and the anion nature) and the temperature of equilibrium were experimentally addressed. In almost all examples it was observed a preferential concentration of caffeine in the polymer-rich phase whereas nicotine and xanthine preferentially migrate to the (opposite) IL-rich phase. In spite of the alkaloids chemical similarities, the studied ABS presented selectivity values of xanthine vs. caffeine as large as 19. The gathered results show that polymer-IL-based ABS allow the selective separation of similar structures by an adequate manipulation of the IL chemical structure and temperature of equilibrium, and can be envisaged as potential platforms to be applied in countercurrent chromatography.