Glycogen synthase kinase-3 (GSK-3) is a potential drug target for a number of human diseases. Some indigoids have been found to be potent inhibitors of GSK-3, and individual compounds with better activity, specificity, and solubility are desired. In this work, a new disubstituted indigoid generation system was developed with a tryptophanase-deficient Escherichia coli strain as a host to express the human cytochrome P450 2A6 mutant L240C/N297Q, which catalyzes the oxidation of indole to isatin and indoxyl, which in turn react to generate indigoids. Forty-five substituted 1H-indoles from commercial sources were used as substrates in the system, and indigoid mixtures were tested as potential inhibitors of GSK-3. After preliminary screening, cell extracts with high inhibitory activity towards GSK-3alpha/beta were fractionated, and the IC50 values of twelve individual indigoids were measured for GSK-3alpha/beta as well as the protein kinases CDK1/cyclinB and CDK5/p25. Several indigoids, including an indigo, showed stronger inhibition than found in previous work. The most potent towards GSK-3alpha/beta, dimethyl indirubin 5,5'-dicarboxylate (IC50 of 51 nM), was modified by chemical reactions. One product, indirubin 5,5'-dicarboxylic acid 5-methyl ester, inhibited GSK-3alpha/beta with an IC50 of 14 nM and selectivity nearly 40-fold over CDK1 and CDK5. Indirubin-5-5'-dicarbonitrile was also modified to the corresponding 3'-oxime, which had low specificity but showed very high inhibition of all three kinases with IC50 values of 5, 13, and 10 nM towards GSK-3alpha/beta, CDK1, and CDK5, respectively. Thus, this system has the potential to generate new indigoids with therapeutic potential.