Due to their ability to metabolize xenobiotics, glutathione S-transferases (GSTs) play an important role in cellular protection. GST family members mu (GSTM1) and theta (GSTT1) exhibit a common polymorphism that results in the complete deletion of the gene (null allele). Homozygous deletions, which result in the absence of the enzyme, are considered a risk factor for several diseases, including cancer. We report a simple, low cost, and high throughput assay for the simultaneous analysis of the GSTM1 and GSTT1 null polymorphisms in a single step. The assay is based on multiplex real-time PCR in the presence of SYBR Green I and genotype discrimination by melting curve analysis in a LightCycler. We have genotyped 792 samples to compare this new approach with conventional PCR followed by gel electrophoresis. Comparison of the methods gave a good agreement, with kappa values of 0.88 for GSTM1 and 0.64 for GSTT1. Reanalysis of discrepant samples indicated that absence of amplification of the larger GSTT1 fragment by conventional PCR accounted for most of the discrepancies. Moreover, the improved amplification efficiency of the real-time PCR results in a significant reduction of missing values. Due to its simplicity and low cost, this assay is well suited for the rapid analysis of GST-null genotypes in studies that involve large number of samples.