Given the biotechnology advances observed in recent years in terms of upstream, the development of effective downstream processes becomes mandatory to decrease the associated costs of biotechnological‐based products. Although a large interest has been devoted to ionic‐liquid‐based aqueous biphasic systems (IL‐based ABS) as tailored separation platforms, imidazolium‐based ILs have been the preferred choice as phase‐forming agents. To overcome some toxicity and biodegradability issues associated to imidazolium‐based ILs, novel ABS composed of ILs analogues of glycine‐betaine (AGB‐ILs) are here proposed and investigated. Five AGB‐ILs were synthesized, characterized in terms of ecotoxicity, and applied toward the development of novel ABS formed with Na2SO4. Three commercial ILs were also investigated for comparison purposes. The respective ABS ternary phase diagrams, as well as the tie‐lines and tie‐line lengths, were determined at 25°C. Finally, their performance as extraction strategies was evaluated with five amino acids (L‐tryptophan, L‐phenylalanine, D‐phenylalanine, L‐tyrosine and L‐3,4‐dihydroxyphenylalanine/L‐dopa). In all studied systems amino acids preferentially migrate to the IL‐rich phase, and with AGB‐ILs, the amino acid extraction efficiencies to the IL‐rich phase range between 65% and 100%, obtained in a single‐step. Furthermore, the studied AGB‐ILs display a higher ability to form ABS and to extract amino acids than ABS composed of more traditional and commercial ILs. In summary, novel ABS composed of AGB‐ILs can be formed and used as separation routes of value‐added compounds of biotechnological interest.