Plant-derived compounds have been recognized by the feed industry as important supplements for livestock welfare and health. In this context, Citrus aurantium L. extract and Origanum vulgare L. essential oil have been demonstrated to have strong anti-inflammatory and antioxidant effects on animals. Being the composition of plant-derived extracts extremely influenced by the environmental and growing conditions of the plants, quality control is necessary in terms of the concentration of the active compounds to assure the reproducibility of natural feed additives. The present work aimed at the validation of the extraction procedure from feed additives of Hesperidin (HES) and Carvacrol (CAR), the main active compounds of Citrus aurantium and Origanum vulgare extracts. Then, the quantification method of both the analytes was developed and validated by reversed high-performance liquid chromatography coupled with a UV detector. The validated method was tested on premixtures and final feed additives supplied by a local feed factory to supervise the production chain. The extraction method with methanol resulted to be efficient and highly reproducible, with recovery higher than 90% for both the analytes. The chromatographic method has been demonstrated to be accurate, precise (relative standard deviation percent lower than 2.06%), and linear in the tested range concentrations, with regression coefficients equal to 0.995 and 0.999 for HES and CAR respectively. The method demonstrated that the feed additives prepared by the factory by diluting the premixtures were less concentrated than what was declared on the label
