Penicillin G acylase (PGA) is one of the most important enzymes for the production of semi-synthetic beta-lactam antibiotics and their key intermediates. Purification of penicillin G acylase from fermentation broth with the aid of high-throughput screening (HTS) process has been examined in this study. We used a microtiter-plate based on screening method to find appropriate purification conditions for the target protein. The screening method is based on a 96-well plate format, and different matrices and conditions (pH, salt concentration and type) were tested. Through analyses of all pooled fractions (flow-through and elution) we gained appropriate information to choose the best performing matrix and buffer conditions for upscaling. After an upscaled purification step the second unit operation is screened in the similar way and parameters for this operation can be chosen. The purification parameter of purified PGA at the small-screen and upscaling levels were measured, respectively. The results indicate that high-throughput progress based on a 96-well plate is a flexible and efficient paradigm for recombinant protein purification. (c) 2008 Published by Elsevier B.V. on behalf of Taiwan Institute of Chemical Engineers.