To achieve standardized and parallelized microscale protein purification, an automated purification system was developed and optimized using a pipetting robot for purifying large sets of proteins with NI-NTA magnetic agarose beads. Recombinant hexahistidine-tagged proteins beta-glucanase (Bgl-His) and esterase I of Pseudomonas fluorescens (PFE I) were tested on the system. High purity along with high coherence was achieved. The purification procedure can be applied to 96 samples simultaneously and only takes about 1/3 of the time required for purification conducted by hand. Due to the stability of the robot system, the consistency of the results is much better than the results achieved through manual purification. This rapid and reliable method is reproducible and applicable to high throughput screening of huge amounts of samples.