The separation of itaconic acid (0.2 mmol-kg(-1)) from its aqueous solution is performed by dissolving tri-n-octylamine (TOA) in the biocompatible mixtures of 1-decanol + n-dodecane, dodecanol + n-dodecane, and oleyl alcohol + n-dodecane at different compositions. Also, the physical extraction data are presented with dodecane, oleyl alcohol, dodecanol, and 1-decanol. Here, fixing the TOA composition at 20 vol %, the vol % of modifiers (oleyl alcohol, dodecanol, and 1-decanol) is changed from 20 to 60 vol % in dodecane (diluent) to find the best combination for the extraction of the acid. The extraction system comprised of 20 vol % TOA + 40 vol % dodecanol + 20 vol % n-dodecane gives the best separation efficiency (95.06%). Then, with this solvent system, equilibrium studies are carried out at different acid concentrations (0.05 and 0.251 mmol.kg(-1)). Based on the theoretical study, the insights of the extraction mechanism are determined in terms of equilibrium constants (overall and individual, K-E, K-11, and K-21) and stoichiometry (m and n) at 298 K.