Abstract: 17α-Hydroxyprogesterone (17-OHP) is a specific diagnostic marker for the screening and therapeutic monitoring of congenital adrenal hyperplasia (CAH). Its accurate measurement is crucial for the early diagnosis and treatment of the disease. The evaluation of measurement uncertainty is an indispensable component of quantitative analysis. This study builds upon a previously established reference method for 17-OHP using isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS). By constructing a mathematical model and following the (ISO/IEC Guide98-3: Guide to the Expression of Uncertainty in Measurement, GUM: 1995), we systematically assessed the uncertainty associated with the measurement of serum 17-OHP using isotope dilution mass spectrometry. The uncertainty of quantitative analysis of serum 17α-hydroxyprogesterone stems from multiple links in the measurement process, mainly including the preparation of the standard curve, sample reconstitution, and the pipetting process, which involve uncertainties from instruments such as balances, volumetric flasks, pipettes, and densitometers. Additional uncertainties arise from factors such as inter-bottle variability, temperature fluctuations during reconstitution, repeat measurements, calibrator purity, and linear fitting. After calculating the uncertainty of each component based on the mathematical model, all components were combined to determine the combined standard uncertainty and expanded uncertainty. This study provides a reference for uncertainty evaluation in similar methods in clinical laboratories.