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Glutaric acid is an attractive chemical compound which can be used for the manufacturing of polyesters, polyamides, and polyols. It can be produced by the synthesis (chemical method) and fermentation (biological method) process. Glutaric acid is presented with the lowest quantity in the fermentation broth and industrial waste streams. The separation methods of glutaric acid are difficult, costly, and non-environment friendly from fermentation broth. Reactive separation is a simple, cheapest, and environment-friendly process for the recovery of carboxylic acid. Which can be employed for the separation of glutaric acid with lower cost and environment-friendly process. In this study, response surface methodology (RSM) was used as a mathematical technique to optimize and experimental design for investigation of the reactive separation of glutaric acid from the aqueous phase. As per RSM study, 20 experiments with different independent variables such as concentration of glutaric acid, % v/v of trioctylamine, and pH for recovery of glutaric acid were performed. The optimum condition with maximum efficiency (η) 92.03% for 20% trioctylamine and pH = 3 at 0.08 mol/L of glutaric acid initial concentration were observed. The lower concentration of trioctylamine provides sufficient extraction efficiency of glutaric acid. This method can also be used for the separation from fermentation broth because a lower concentration of trioctylamine which makes this process environment-friendly. The optimization condition-defined quadratic response surface model is significant with R 2 of 0.9873. The independent variables defined the effect on the extraction efficiency of glutaric acid. This data can be used for the separation of glutaric acid from industries waste and fermentation broth.