Clinical studies have suggested that a defect in both glutathione S-transferase (GST) M1 and GSTT1 increases the risk of drug-induced hepatotoxicity. The present study developed the method that enables genotyping of GSTM1 and GSTT1 directly using a small aliquot of blood samples based on an isothermal Smart amplification process version 2 (SmartAmp-2). SmartAmp-2 reaction could complete the genotyping of GSTM1 and GSTT1 within 40 min. The frequency of wild-type, GSTM1 null, GSTT1 null, and double null was 24, 21, 35, and 19%, respectively, consistent with previous reports in the Japanese population. The genotypes of 94 human genomic DNA samples determined by SmartAmp-2 were identical to those determined by the conventional polymerase chain reaction method. SmartAmp-2 was able to determine the genotypes of GSTM1 and GSTT1 even when human blood specimens were used. The SmartAmp-2 method is a rapid and accurate means of identifying the GSTM1 and GSTT1 genotypes, making it less time and more labor efficient in clinical practice than conventional methods requiring preparation of genomic DNA and electrophoresis. This will contribute to evaluate the susceptibility of disease and adverse reactions to drugs caused by deletion of GSTM1 and GSTT1.