This study explored the sample preparation steps based on QuEChERS (quick, easy, cheap, effective, rugged, and safe) of a complex matrix, such as sewage sludge, regarding their impacts on the chemical signatures of the samples obtained via high-resolution mass spectrometry (HRMS). The effects on the HRMS signal of the intrinsic parameters of the QuEChERS method, such as the extraction solvent, the buffer salt, or the purification procedure, have been evaluated. Different strategies have been used to perform these evaluations, such as a comparison of the total number of HRMS signals, a comparison of the targeted screening based on more than 100 compounds, and a comparison of a nontargeted screening based both on the study of the most discriminating couples [mass-to-charge ratio (m/z); retention time (tR)] and on the most intense peaks. Moreover, two specific methods dedicated to the extraction of surfactants and drugs have been added to this comparison to visualize the impact of specific conditions on the chemical signature. This study provides the first information on the impact of sample preparation on the HRMS analysis of sludge. It shows that acetonitrile is the most effective solvent for obtaining maximum information from sewage sludge. The comparison of the different cleanup procedures emphasizes the significance of this step and demonstrates the connection between the intrinsic physicochemical properties of molecules and the operating conditions of the extraction, particularly the pH. Moreover, the study highlights that surfactants, i.e., quaternary ammoniums in the positive ionization mode and linear alkylbenzene sulfonates in the negative mode, are the most discriminating compounds.