This paper reports the phase separation conditions for "sugaring out", a new phase partition method. It was observed in our laboratory that the addition of a monomeric sugar or a disaccharide in an acetonitrile (ACN)-water mixture created two phases with the upper phase rich in acetonitrile and a lower aqueous phase. Potential applications of this method have been explored for the extraction of high value products such as biomolecules (proteins and antibiotics), hydroxymethyl furfural, furfural, syringic acid, ferulic acid, and p-coumaric acid from the aqueous phase. However, the detailed phase separation conditions are unknown for this method. Hence, this work was undertaken to study the phase separation conditions for sugaring-out. The effect of temperature and glucose concentration on sugaring-out was investigated. Tie line data of ACN-water-glucose system were measured and satisfactorily correlated with the Othmer-Tobias and Bancroft equations. It was observed that high glucose concentration and low temperatures favor sugaring-out. The advantages of sugaring-out make this method suitable for a large number of applications in the chemical and biotechnology industry.