This study describes the evaluation and optimization of a crystallizing process capable of efficiently purifying vancomycin in high purity and yield. In particular, we observed how the main process parameters influenced the formation of crystals, determined their morphology, and monitored purity and yield. Acetone was shown to be more effective than alcohol solvents for the crystallization of vancomycin. The optimal distilled water/acetone ratio, storage temperature, storage time, pH, conductivity, initial vancomycin concentration and stirrer velocity were shown to be 1 : 3.5 (v/v), 10 ℃, 24 h, pH 2.5, 20 ms/cm, 0.1 g/mL, and 640 rpm, respectively. Temperature had a decisive influence on crystal formation; crystals were successfully produced at 10 ℃, while at other temperatures, conglomeration, disintegration and cohesion occurred. Crystal growth developed over time and was complete at about 24 h. Vancomycin purity remained at about 97.0% irrespective of storage time while the yield increased over time, reaching a maximum of 95.0% at around 24 h, after which there was no substantial change. Crystallization occurred over a certain range of pH (2.5-3.0), but purity and yield were highest at pH 2.5. When the pH was outside this range, a conglomeration (gelation) phenomenon prevented the efficient production of crystals. Vancomycin crystals were produced irrespective of the stirrer velocity, which had no influence on purity; however, the highest yield of vancomycin was obtained at 640 rpm.