Hybrid short path evaporation (HSPE) is proposed as an alternative separation process with potentiality for recovery and concentration of thermally unstable molecules such as lactic acid. This work aimed to analyze the influence of HSPE process variables on the concentration of lactic acid from sugarcane molasses. Through 22 factorial experimental design, the influence of evaporator temperature (from 115 to 135 °C) and internal condenser temperature (from 13 to 22 °C) on lactic acid concentration and mass percentage of residue and distillate stream were studied. The results showed that it is possible to increase the lactic acid concentration greater than twice from fermentation broth by HSPE. In addition, the effect of each variable studied and interactions between them were demonstrated.