tProcedures for estimating the measurement uncertainty (MU) of the concentration of a given analyte ina sample are of major concern for analytical chemists. Unfortunately, it is still unclear how and why MUshould be assessed. While several possibilities exist, an appropriate approach consists in using methodvalidation data for the evaluation of MU. This was demonstrated by a validation study achieved inthe framework of a clinical study related to caffeine in sports medicine, where the results were usedfor the evaluation of MU. After validation of the method developed using ultra-high pressure liquidchromatography–mass spectrometry for caffeine and its three main metabolites, accuracy profiles werebuilt for each analyte. The first important conclusion is that the developed method was valid for allcompounds and met the given specifications for the application (fit for purpose). Relevant estimates ofcombined standard uncertainty were computed to obtain uncertainty functions, which allow obtainingvalues of MU as a function of the concentration of the analyte. The great advantage of both uncertaintyfunction and uncertainty profile is the development of a continuous model that enables easy calculationof the standard, expanded and relative expanded uncertainty at any concentration within the validationdomain. In fact, the expanded uncertainty interval is assumed to contain 95% of all possible measure-ments, regardless of the concentration. Finally, the uncertainty function enables the determination of thelowest limit of quantification by selecting adequate acceptance limits, with the limit of quantificationbeing defined as the point where the relative uncertainty equals the acceptance limit threshold. It hasto be noted that further discussions remain mandatory to establish which criteria should be applied todefine an adequate decision threshold, and the proposal afforded in this work may open new avenues inthis direction.