Background: CYP2B6 is a highly variable and polymorphic cytochrome P450 (CYP) enzyme involved in the biotransformation of an increasing number of drugs, including cyclophosphamide, bupropion, and the nonnucleosidic reverse transcriptase inhibitor efavirenz. Several nonsynonymous and promoter single-nucleotide polymorphisms (SNPs) in the CYP2B6 gene are associated with altered hepatic expression and function, which affect drug plasma concentrations. Methods: We used multiplex PCR to amplify relevant gene fragments while avoiding amplification of the CYP2B7P1 pseudogene. Polymorphic sites were analyzed by allele-specific primer extension followed by matrix-assisted laser desorption/ionization time-off-light mass spectrometry (MALDI-TOF MS). Method evaluation was performed on a panel of 287 genomic DNA samples previously genotyped by other methods. Results: Five multiplex assays were developed, comprising the following 15 SNPs: -82T [right arrow] C (* 22); 86G [right arrow] C (R29T, * 17); 136A [right arrow] G (M46V, * 11); 296G [right arrow] A (G99E, * 12); 415A [right arrow] G (K139E, * 8, * 13); 419G [right arrow] A (R140Q, * 14); 516G [right arrow] T (Q172H, * 6, * 7, * 9, * 13, * 19, * 20), 547G [right arrow] A (V183I); 769G [right arrow] A (D257N); 785A [right arrow] G (K262R, * 4, * 6, * 7, * 13, * 16, * 19, * 20); 983T [right arrow] C (I328T, * 16, * 18); 1006C [right arrow] T (R336C, * 19); 1172T [right arrow] A (I391N, * 15); 1282C [right arrow] A (P428T, * 21); 1459C [right arrow] T (R487C, * 5, * 7). In 9 DNA samples showing discrepant genotypes, correctness of the MALDI-TOF MS result was confirmed by direct sequencing. Conclusions: This genotyping method enabled sensitive, specific, accurate, and comprehensive determination of 15 relevant SNPs of CYP2B6. The assay design allows analysis of SNP subsets, incorporation of additional SNPs, and performance of high-throughput genotyping.