We developed a novel method of producing proteins containing multiple disulfide bonds in a cell-free protein synthesis system. To provide an optimized redox potential during the synthesis of truncated plasminogen activator (rPA), we pretreated the E. coli S30 extract with an excess amount of oxidized glutathione based on the anticipation that the reducing potential of the S30 extract would be exhausted through the reduction of the oxidized glutathione molecules. As expected, it was found that the reducing activity of the S30 extract was remarkably decreased through the pretreatment, and active rPA was produced when the pretreated S30 extract was used after removing the residual glutathione molecules. In particular, compared to the method involving the iodoacetamide treatment of S30 extract, the present protocol was effective in producing active rPA during the batch reaction of cell-free protein synthesis.