Description:The misuse of anabolic androgenic steroids in livestock remains a major concern for food safety and regulatory monitoring, as exogenous administration of endogenous hormones such as testosterone is difficult to confirm using conventional analytical approaches. This project aimed to develop an untargeted metabolomics workflow based on ultra-high-performance liquid chromatography coupled to high-resolution Orbitrap mass spectrometry (UHPLC-Q-Orbitrap-MS) to investigate metabolic alterations in cattle serum following testosterone propionate administration. The study focused on optimizing data acquisition and processing strategies, including feature detection, alignment, and statistical evaluation using multivariate and univariate approaches. The resulting workflow provides a comprehensive analytical framework that can support future regulatory and research applications related to hormone misuse detection in food-producing animals.