Differential gene expression profiling has become of central importance for the analysis of cellular systems at the transcriptional level. By now, many platform technologies including DNA-microarrays, serial analysis of gene expression or RNA-seq have been established in order to facilitate transcriptional profiling. However, these technologies are all subjected to specific limitations, as they require a priori knowledge of annotated genome sequences or are based on substantial bioinformatic infrastructure, for example. As an unbiased alternative we describe here a multidimensional electrophoretic system of separation for the analysis of gene expression for the global transcriptional profiling in any eukaryotic organism. This approach is compatible with standard laboratory equipment comprising high-resolution separation of complex cDNA-probes using two-dimensional DNA-gel electrophoresis. In this context cDNA fragments are separated using non-denaturing PAGE in the first dimension with subsequent denaturing gradient gel electrophoresis in the second dimension. Two-dimensional spot patterns are quantified by well-established bioinformatic algorithms and selected spots are identified using DNA sequencing. Neither does this method necessarily depend on annotated genome sequences, nor does it require sophisticated instrumentation. Strikingly, quantitative data on differential gene expression derived from multidimensional electrophoretic system of separation for the analysis of gene expression highly correlate with corresponding data from quantitative RT-PCR even for transcriptional profiles of limited amounts of total RNA.