Objectives: Deletion polymorphisms of Glutathione-S-transferase (GST) M I and T I are considered risk factors for various diseases. However, most previous studies only distinguished "null" and "non-null" genotypes. Our aim was to develop a reliable. high-throughput GSTM1/T1 genotyping method able to determine allele copy numbers. Design and methods: We developed a multiplex real time PCR method to distinguish between heterozygous (1/0) and homozygous (1/1) GSTM1 and GSTT1 genotypes. The principle of relative quantification was applied and an expectation-maximisation (EM) algorithm was developed to assign one of 3 possible genotypes: 1/1, 1/0 or 0/0 for each of the two genes. Results: 1320 Caucasians were genotyped using the newly developed method. The observed genotype distributions did not deviate from the expected and were in Hardly-Weinberg equilibrium. GSTM1 duplication was detected in one sample. Conclusion: This new semiquantitative genotyping method is a sensitive and promising tool for large-scale molecular epidemiological and clinical studies.