Vascular lesion formation and collagen presentation are key events leading to the development of vulnerable plaques. Glycoprotein VI (GPVI) significantly contributes to plaque-associated collagen binding and thrombus formation. The aim of this study was to image endothelial injury using fluorescence-labelled GPVI-Fc (Fc, fragment crystallized), a soluble form of GPVI that was generated by cloning and fusing GPVI to an Fc-domain, in an ex-vivo rabbit model. This study serves as a proof-of-principle study to demonstrate that GPVI-Fc is a useful tool for detecting endothelial damage. The carotid and femoral arteries and the aorta abdominalis were isolated from rabbits and perfused with phosphate buffered saline (PBS) to remove all blood, and a catheter was placed into the vessels in situ. Endothelial damage was achieved by pulling an inflated balloon approximately 1 inch through the vessels, while control vessels were not balloon-treated. After balloon deflation, the catheter was removed. Fluorescence-labelled GPVI-Fc (50 µg/mL) was injected into the injured and control intact vessels, and the opened vessels were sealed by clamps. After incubation, the vessels were rinsed with PBS, and optical imaging was performed to measure GPVI-Fc binding to injured endothelium. The optical data corresponding to the mean detected optical signal of the regions of interest were corrected by subtracting the mean data of the background fluorescence (arbitrary units). After denudation, fluorescence was enhanced in injured femoral and carotid arteries when compared to intact femoral (41.1 ± 17.5 vs. 14.6 ± 6.5; P = 0.021) and carotid (30.2 ± 7.6 vs. 7.9 ± 3.9; P = 0.005) arteries. This preclinical GPVI-Fc-based vascular lesion imaging approach may be the first step towards a method that allows identification of vascular lesions in vivo.