The O(6)-methylguanine DNA methyltransferase (MGMT) gene encodes a DNA repair enzyme whose activity is a major mechanism of resistance to alkylating drugs in glioblastoma treatment. Hypermethylation of the MGMT promoter is associated with chemosensitivity because it reduces MGMT activity. Here we present a method combining methylation-specific and SYBR-green-based quantitative PCR (MSQP) for MGMT promoter methylation analysis. This highly specific, sensitive, and reproducible method allows the quantification of fully methylated and fully unmethylated MGMT DNA species in terms of percentage. Values are related to standard curves, corrected for DNA input by an internal standard, and calculated in relation to methylated and unmethylated control DNAs as a percentage share. Finally, values are defined relative to the sum of fully methylated and unmethylated MGMT DNA sample amount to obtain percentage of methylated reference and percentage of unmethylated reference results. We have used this technique to investigate MGMT promoter methylation in relation to MGMT mRNA expression in nine tumor cell lines and 15 primary glioblastoma patients. Presented data confirm that this assay is suitable for detection of low amounts of methylated and unmethylated MGMT promoter DNA. Carefully validated quantitative MSQP assays will be useful in both research and clinical molecular diagnosis.