This study was conducted to establish age‐related reference ranges for two heparin‐binding proteins – vitronectin and platelet factor 4 (PF4) – and to determine if the quantitative values of these proteins may contribute to the reported age‐dependent effect of unfractionated heparin (UFH). Plasma samples were obtained from healthy children aged between 1 month and 16 years and from healthy adult volunteers. Two commercial kits were used to measure plasma vitronectin and PF4 levels. Results were reported as mean and boundaries including 95% of the population. Plasma vitronectin levels for children aged 1–5 years were significantly higher compared with adults. Plasma PF4 levels for infants <1 year of age were significantly lower compared with adults. The differences between reference values for both proteins in all other age‐groups were not statistically significant. This study for the first time has established age‐related reference ranges for vitronectin and PF4. In establishing these ranges, the quantitative values of these proteins do not appear to be the major contributory cause for the age‐dependent variation in UFH effect. Future studies are required to evaluate the possible impact of age‐dependent differences in binding between heparin‐binding proteins and UFH.