Data from comprehensive two-dimensional (2-D) separation techniques, such as comprehensive 2-D gas chromatography (GC x GC), liquid chromatography/liquid chromatography (LC x LC) and liquid chromatography/ capillary electrophoresis (LC x CE) can be readily analyzed by various chemometric methods to increase chemical analysis capabilities. A retention time alignment, preprocessing method is presented that objectively corrects for run-to-run retention time variations on both separation dimensions of comprehensive 2-D separations prior to application of chemometric data analysis algorithms. The 2-D alignment method corrects for run-to-run shifting of a sample data matrix relative to a standard data matrix on both separation time axes in an independent, stepwise fashion. After 2-D alignment, the generalized rank annihilation method (GRAM) is successfully applied, substantiating the performance of the alignment method. The alignment method should have important implications, because most 2-D separation techniques exhibit, in the context of chemometric data analysis, considerable run-to-run retention time shifting on both dimensions. Even when there are only three to four points/peak, that is, with three to four separations on the second dimension (column 2) per peak width from the first dimension (column 1), the 2-D alignment coupled with GRAM provides dependable analyte peak identification capabilities and adequate quantitative precision for unresolved analyte peaks. Thus, the 2-D alignment algorithm is applicable to lower data density conditions, which broadens the scope of chemometric analysis to high-speed 2-D separations.