The most important source of Salmonella spp. infection in humans is by the consumption of contaminated poultry products. Due to the risk of resistance development and its transfer from animals to humans, the Belgian Royal Decree concerning the eradication of Salmonella (C-2007/22784) prohibits treatment of poultry with antimicrobials against zoonotic Salmonella spp. To uncover illicit use, an analytical method using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) for the determination of antimicrobial residues in poultry excreta was developed and validated for classes having an active spectrum against Salmonella spp. in poultry: β-lactams (amoxicillin and penicillin V), fluoroquinolones (enrofloxacin, difloxacin, and flumequine), polymyxins (colistin), sulfonamides in combination with trimethoprim (sulfachloropyridazine, sulfadiazine, and sulfaclozine), and tetracyclines (chlortetracycline and doxycycline). A generic and high-throughput sample preparation was developed. Extraction of samples was performed by ultrasonication using a combination of acetonitrile and McIlvaine buffer, followed by centrifugation and filtration prior to analysis. The method was validated according to Commission Decision 2002/657/EC for linearity, apparent recovery/trueness, repeatability, reproducibility, limit of quantification, limit of detection, specificity, matrix effect, and storage stability in matrix. To demonstrate the applicability of the method, an in vivo experiment was conducted. For each antimicrobial class, one registered drug was selected and administered in the drinking water to two laying hens. Excreta samples were collected every 12 h during and until 2 days after treatment and analyzed using the developed method.