Amino acids are important in human and animal diet, as well as being potential feedstocks for chemical production. Amino acids can be obtained from protein after hydrolysis. In addition, several agro-industrial residues already contain a mixture of free amino acids. The objective of this study was to develop a method for amino acids separation, starting from mixtures containing amino acids, and using antisolvent precipitation with ethanol. Protamylasse, rubber seed protein hydrolysates, and grass juice were used in the experiments, representing existing and potential agro-industrial residues. Our results show that in a water-ethanol system, some amino acids had lower solubility in mixtures than as a single component, thereby facilitating precipitation. A sufficiently high total amino acid concentration in the mixture is needed to achieve precipitation; therefore, a concentration step is sometimes required. Ethanol precipitation can be applied as a pretreatment to separate mixtures into groups of amino acids or a polishing step to increase purity.