The development of a generic analytical method remains difficult when a high number of compounds has to be simultaneously considered. This study proposes an innovative strategy for the development of a solid phase extraction (SPE) procedure before liquid chromatography-mass spectrometry analysis of 34 diuretics and beta-blockers in urine samples. These compounds have been selected since they are often encountered in anti-doping control. The principle is based on the selection of representative analytes during SPE protocol optimization, allowing a drastic reduction of generated data and development time. To select the representative compounds, all substances were classified based on their SPE behavior with a generic method and groups were formed with the help of a chemometric tool, namely hierarchical cluster analysis (HCA). One representative analyte per group was selected and used for subsequent SPE method development. Once the SPE method was developed, compounds were analyzed by LC-MS and matrix effects were evaluated to determine the influence of the matrix on the SPE process and MS signal alteration due to endogenous compounds. As a result, matrix effects evaluation must be performed on all analytes; representative compounds previously selected for SPE development were unable to predict matrix effects.