Current clinical recommendations for unfractionated heparin (UFH) therapy suggest target APTT ranges should reflect heparin concentrations of 0.2-0.4 IU/ml by protamine titration or 0.35-0.7 IU/m by an anti-Xa assay. Historically, performance of a manual protamine titration assay has been labour intensive and required a large plasma sample. However, recent studies have described difficulties with standardizing anti-Xa assays and demonstrated poor correlation of anti-Xa assays in children. This study aimed to refine and test the feasibility of a modified protamine titration assay using 100 microl of plasma. The resultant method produced reliable and repeatable results in adult plasma pools spiked with UFH. The feasibility of this method was proven by testing of in vivo heparinised samples obtained from children. This protamine titration method may offer an alternative to anti-Xa assays for clinical monitoring of children on heparin therapy, and will enhance clinical studies investigating paediatric-specific management of UFH therapy.