Biopharmaceuticals are commonly present in relatively low concentrations in aqueous solutions, making their detection and purification detrimental. In this work, we used novel aqueous biphasic systems based on glycerol formal (GF) to extract an important antibiotic - tetracycline. We report cloud points (solubility curve) and tie-lines for three ternary systems, containing GF, water, and inorganic salt (either K ₃PO ₄, K ₂HPO ₄, or K ₂CO ₃) at constant temperature of 298 K and at 0.1 MPa. The tie-line data of these ternary systems were correlated using the nonrandom two-liquid model, and binary interaction parameters of activity coefficients were estimated. The experimental and correlated tie-line data were compared in terms of average root-mean-square deviation and showed satisfactory agreements. The partition coefficients of tetracycline between two phases were measured, and corresponding extraction efficiencies were calculated. The maximum value of partition coefficient was 1551 for the system containing K ₃PO ₄, followed by values of 1145 and 927 for systems containing K ₂CO ₃ and K ₂HPO ₄, respectively. The calculated extraction efficiencies were very high - greater than 98.8%, demonstrating high potential for using aqueous biphasic systems based on GF for separation and purification processes.