Abstract: If testosterone in food reaches the limit critical value of 2.0 μg/kg, uncertainty assessment is necessary to enhanceaccuracy and food qualification judgment. We conducted an uncertainty assessment based on the methods outlined in Announcement No. 1031-1-2008 of the Ministry of Agriculture of China on the detection of 11 hormone residues in animal-derived foods by liquid chromatography-tandem mass spectrometry (LC-MS/MS), the Evaluation and Expression of Uncertainty in Measurement (JJF1059.1-2012), and the Evaluation of Uncertainty in Chemical Analysis (JJF 1135-2005). The analysis was performed using an ultra-performance liquid chromatography-tandem mass spectrometer with isotope internal standardization for quantification. A mathematical model was established to analyze the sources of uncertainty, and an uncertainty assessment was conducted for the determination of testosterone content in goat meat. The assessment results showed that when the measured testosterone content in goat meat was exactly 2.00 μg/kg, its relative combined standard uncertainty was 4.495×10⁻² μg/kg, and the expanded uncertainty was 0.18 μg/kg (confidence level of 95%, k=2). The contribution rates of various uncertainty components, in descending order, were standard solution preparation, sample measurement, standard curve fitting, recovery of spiked blank samples, and sample pretreatment. Therefore, when the detected testosterone content exactly equals the limit, it cannot be directly determined whether it is qualified or not. Instead, more precise pipettes should be used for standard solution preparation, a series of standard working solutions tailored to the sample content should be prepared, and the number of parallel measurements should be increased. The major contributors to uncertainty are standard solution preparation, standard curve fitting, and sample measurement. Thereby ensuring the accuracy and reliability of the measurement results.