There is substantial evidence for a causal relationship between genetic variability of the CYP2D6 gene and changes in the pharmacokinetics of drugs. Therefore, knowledge of single-nucleotide polymorphisms (SNPs) prior to drug administration is highly desired for assisting in the development of individualized pharmacotherapy. We therefore developed a robust assay that detects common CYP2D6 alleles within 60 minutes of blood withdrawal and links carriers of the variant CYP2D6*3 and *4 alleles to the pharmacokinetics of tramadol. This new genotyping assay employs fluorescence resonance energy transfer (FRET) analysis, which permits parallel identification of the CYP2D6*3 and CYP2D6*4 alleles within 60 minutes of blood withdrawal. We determined the genotypes of 100 healthy unrelated individuals and studied the pharmacokinetics of tramadol in 24 CYP2D6 genotyped healthy subjects. The total allelic frequencies of homozygote carriers were 0.015 and 0.25 for the CYP2D6*3 and *4 alleles, respectively, and the plasma area under the curve (AUC) was 84% above those of extensive metabolizers (homozygous EM group): 3,941.2 ng/mL.h (95% confidence interval [CI], 2,928.9 ng/mL.h to 4,953.5 ng/mL.h) versus 2,142.6 ng/mL.h (95% CI, 1,829.6 ng/mL.h to 2,455.7 ng/mL.h). Likewise, the AUC for the O-desmethyl-tramadol metabolite (M1) was significantly reduced in poor metabolizers (PMs): 300.2 ng/mL.h (95% CI, 260.3 ng/mL.h to 340.0 ng/mL.h) versus 842,6 ng/mL.h (95% CI, 715.1 ng/mL.h to 970.0 ng/mL.h). We observed a statistically significant correlation between plasma tramadol AUC and production of the O-desmethyl metabolite in CYP2D6 genotyped healthy volunteers. Our assay can be used reliably in clinical pharmacology studies and may be used for dose adjustment.