von Willebrand Factor (vWF) is a multimeric plasma protein important for platelet plug formation. As part of its haemostatic role, it is released from endothelial cells during vascular stress or injury and is considered an excellent biomarker of endothelial function. Currently, there are no validated kits available to measure vWF in rabbits. We developed a sensitive and reproducible sandwich enzyme-linked immunosorbent assay (ELISA) for detection of vWF in rabbit plasma using commercially available antibodies and reagents. Purified human vWF was used as a calibrator standard with a dynamic range of 1.56–100 ng/mL. The Minimum Required Dilution for rabbit plasma was 1:100. When plasma was spiked with 3.76 or 10 ng/mL vWF, recovery was 108 ± 2% and 93 ± 2%, respectively. Intra- and inter-assay precision for 8 rabbit plasma samples were 3% and 4%, respectively. The Minimum Detectable Concentration was 254 pg/mL for purified human vWF and 1:10,700 dilution of cholesterol-fed rabbit plasma, and the Reliable Detection Limits were 457 pg/mL and 1:5940. Three freeze-thaw cycles significantly decreased vWF concentrations for purified human vWF and 2 of 3 plasma samples assayed. This ELISA provides sensitive and reproducible measurements of rabbit plasma vWF, which is an important biomarker for cardiovascular research.