Disorders of haemostasis result in both excessive bleeding and clotting and are a major global cause of morbidity and mortality, particularly in the developing world. A small number of simple tests can be used to screen and monitor for such dysfunctions, one of which is the prothrombin time (PT) test and associated International Normalisation Ratio (INR). PT/INR is routine in hospital laboratories in developed countries, and can also be performed using point-of-care instruments. However, neither of these approaches is appropriate in low-resource settings. Significant interest has grown in paper-based devices to form the basis of simple and low-cost assays that may have the potential for application in such environments. This study describes the development of a simple, low-cost, paper-based lateral flow prothrombin assay. The assay employed wax printing on chromatography paper to define test channels, with deposition of thromboplastin reagent and calcium chloride onto the resulting strips. These were placed in a test housing and measurement of the flow rates of deposited plasma samples were performed in triplicate. The flow dynamics of the assay was optimised according to the type of paper substrate used, the nature and quantity of the thromboplastin reagent, the amount of calcium chloride required, and the volume of sample employed. An optimised assay configuration demonstrated a dynamic range of 6 mm between normal and factor-deficient plasmas. The assay showed good correlation with laboratory-based PT assay (Yumizen G200) in artificial plasmas in the 9.8 to 36 s range (r2 = 0.8112). The assay also demonstrated good dynamic range and correlation in patient plasma samples in comparison with hospital PT, with a range of 9.8 to 45 s (r2 = 0.7209).