Mupirocin (a mixture of A, B, C, and D pseudomonic acids-four complex, similar structures) is an antibiotic belonging to the monocarboxylic acid class, used to cure infections caused by bacteria (Gram-positive), especially methicillin-resistant Staphylococcus aureus (MRSA). Its biotechnological production and separation need constant attention, as improving antibiotics is important in the pharmaceutical industry. This is the first study regarding pseudomonic acids separation through reactive extraction; the experiments were focused on finding a proper combination of extractant and diluent for optimization of the separation yield. The pH influence (4-9) and extractant (TOA and Amberlite LA-2, 5-20 g/L) concentration dissolved in n-heptane (green solvent) have been analyzed, obtaining the maximum extraction yield (88.78%) at pH 4, 20 g/L extractant, and 10% octanol added to the organic solvent. The addition of the phase modifier, 1-octanol, improved the extraction yield by 2.6 times for Pseudomonic acid A due to interactions of formed complexes with the phase modifier that improves its solubility. The experimental results for determining the mechanism of the interfacial reaction showed that, regardless of the pH value and solvent polarity (modified by the addition of 1-octanol), only one molecule of Pseudomonic acid A and extractant react at the aqueous-organic interface. In addition, the system was modelled and optimized with a methodology combining artificial neural networks (ANN) and differential evolution.