Many assay systems for D-dimer measurement are available. Their intended use is mainly the exclusion of venous thromboembolism (VTE). Despite standardization attempts, an important variability in assay results is observed, and therefore data obtained by use of one assay cannot be extrapolated to another assay. As a consequence, each assay must be validated in appropriate clinical trials to determine its cut-off value for VTE exclusion. The differences observed can be explained by the heterogeneity of fibrin degradation products present in patient samples, by the reactivity of the various antibodies and their combinations, and by the differences in calibrators and in the format of assays. Among the different assay systems available, the use of automated, observer-independent tests having good analytical sensitivity is highly recommended. The assay should also exhibit a high sensitivity for VTE exclusion to be used as the first step of any diagnostic strategy.