The functional characterization of single nucleotide polymorphism (SNPs) represents a major challenge for pharmacogenetics and related research areas. Here, we propose a procedure, termed mRNA expression imbalance assessment, that can be applied to detect cis-acting SNPs with an effect on mRNA expression. The procedure is based on the observation that the relative transcript levels derived from the two alleles of an autosomal gene are reflected in the sequence of the amplified cDNA. The key element of the procedure is the detection of a discrepancy between the specific nucleotide signal intensity of a marker SNP in the genomic DNA and in the cDNA. We used the CYP3A5*1/*3 polymorphism as a proof principle for this approach. In this case, we could even demonstrate that the procedure works equally well with the appropriate tissue samples and in silico, using the existing databanks of sequences. In conclusion, the procedure provides a fast and easy tool, which may facilitate identification of functional SNPs.