Two ethyl lactate (EL)-based aqueous two-phase systems (ATPSs) were studied at T = 298.15 K and atmospheric pressure. Sodium tartrate and potassium sodium tartrate organic salts have been used to form biphasic systems. To assess the phase splitting, binodal curves and tie-lines were obtained experimentally by the cloud point method and a technique, which consist of the density and refractive index, respectively. Binodal curves were fitted with the Merchuk equation obtaining a good fit. Moreover, modeling with the universal quasichemical thermodynamic equation was carried out to accurately represent the two-biphasic systems. Furthermore, experimental binodal curves obtained in this work and literature results for the same systems and for systems with EL and other salts were analyzed regarding their similarity. Finally, the application of the adopted ATPS in the partitioning of two flavonoids, rutin and quercetin, was studied in terms of the partition coefficients and the percentage of extraction. Rutin and quercetin showed better affinity to the EL-rich phase, evidencing that the use of the proposed ATPS may be a successful technique in the biotechnological industry for the separation of each flavonoid.