Glatiramer acetate (GA), FDA-approved therapeutic for multiple sclerosis (MS), is a random-sized synthetic polypeptide composed of four amino acids: tyrosine, glutamic acid, alanine, and lysine. This work aims to design an efficient protocol for the separation of GA from its broth. For this purpose, the feasibility of a carbohydrate-based aqueous two-phase system (ATPS) for separation of GA from the amino acids was investigated. Initially, the binodal curve and tie-lines for the ATPS composed of maltodextrin and acetonitrile were determined. Next, preliminary screening experiments for determining optimum conditions were conducted by introducing GA and amino acids into the ATPS individually and the effect of pH and carbohydrate concentration on partition coefficient were investigated. Results revealed that glutamic acid and alanine were migrated toward the acetonitrile rich phase while GA, tyrosine, and lysin showed the opposite trend. Besides, the partitioning of amino acids was correlated with their physicochemical and structural properties. The preliminary results suggested that the system composed of maltodextrin 15 wt%, acetonitrile 35 wt%, at pH = 6 can be considered as the optimum feed. In the next step, the partitioning of GA and amino acids broth was investigated in the optimum feed. Under these conditions, the partition coefficient and selectivity of GA were obtained to be 1.17 and 62%, respectively. Additionally, circular dichroism spectroscopy results proved that the structure of GA remains unchanged during the separation steps. Finally, a recovery method was proposed where the acetonitrile was evaporated and maltodextrin was precipitated by the addition of methanol.