ABSTRACT In animal husbandry for food production, the administration of non-steroidal anti-inflammatory drugs (NSAIDs) as anti-inflammatory, analgesic, and antipyretic agents, has seen an increase due to raised awareness of animal welfare issues. Residues of NSAIDs may be present in animal products that are intended for human consumption, and since some may pose a certain risk to human health, there is a need to monitor NSAID residues at low levels via routine and targeted surveillance. In analytical chemistry, NSAIDs are usually differentiated using their acid-base properties. Within this study, a method for simultaneous analysis of 27 NSAIDs, including both groups, in milk and muscle samples in 12.5 min is described. Sample processing consisted of enzymatic hydrolysis and acetonitrile extraction, followed by a clean-up of the extract by SPE, and measurement by UHPLC-MS/MS. The in-house validation study (alternative approach), covering trueness, precision, sensitivity, decision limit (CCα), detection capability (CCβ) and matrix effect, was designed and evaluated with the help of validation software to meet the demands of regulatory compliance. The method recovery for milk and muscle matrix was in the range of 98.1% to 106.5% and 98.8% to 102.7%, whereas the CCβ as the parameter for screening analysis, ranged from 0.07 to 46.7 µg/kg and 1.19 to 69.7 µg/kg, and the CCα, as the parameter for confirmatory analysis, from 0.11 to 56.7 µg/kg and 1.12 to 518.6 µg/kg, respectively. The occurrence of NSAID residues in milk and muscle samples was assessed using the developed method within the Croatian National Residue Control Plan, revealing quantifiable residues for diclofenac, ketoprofen, and salicylic acid mostly in milk samples. The most abundant NSAID in analysed samples was salicylic acid, which may be introduced into the food chain and be present in various types of matrices due to its natural occurrence in plants as a phytohormone.