Ionic liquid-based aqueous biphasic systems (IL-ABS) formed by ILs composed of ions of low toxicity, choline ([Chol]+) coupled with saccharinate ([Sac]−) and acesulfamate ([Ace]−), and inorganic salts with distinct water-structuring properties were employed for simultaneous extraction and concentration of acetylcholinesterase (AChE) inhibitors – galantamine (gal), N-desmethyl galantamine (des) and ungiminorine (ung). Comprehensive experiments aimed to assess the influence of salt and IL type and concentration, as well as the pH and temperature on the phase-forming ability and distribution of the target alkaloids between the two phases formed reveled that the IL anion and pH are the most important factors. At the optimal conditions found a quantitative recovery into the IL-rich phase of gal, des and ung was achieved in a single extractive step. These results were further used as a platform for the development of a simple and safer sample pretreatment method for analysis of the three analytes, followed by RP-HPLC/UV detection. The method showed satisfactory analytical performance, the latter allowing quantitative determination of these AChE inhibitors in pharmaceutical dosage form and in human urine.