Lactic acid (LA) is an organic acid produced by fermentation or chemical synthesis. It plays a crucial role in the pharmaceutical, food and plastic industries. In the fermentation of, for example, grass silage, LA and different compounds are produced. To purify lactic acid, researchers have tried to investigate membrane technology to achieve a high yield of lactic acid permeance. This study tested four commercially available nanofiltration membranes (NF270, MPF-36, Toray NF, and Alfa Laval NF). Nanofiltration experiments were performed to investigate the rejection levels of lactic acid from a binary solution by using distinct molecular weight cut off membranes. All of the experiments were conducted with a lab-scale cross-flow membrane unit. Different operating conditions (pH, temperature) were studied for each membrane; the optimal process condition was found at 25 °C and pH 2.8. With higher temperatures and pH, an increase in LA rejection was observed. The MPF-36 membrane shows the lowest lactic acid rejection yield of 7%, while NF270 has the highest rejection yield of 71% at 25 °C and pH 2.8. These results will be helpful in the future to understand both the interaction of lactic acid permeance through nanofiltration membranes and process scale-up.