Thrombosis is an important pathophysiologic component of many cardiovascular diseases. Thrombin, a serine protease, plays a central role in thrombosis formation. Detection and imaging of thrombin activity may thus be of considerable biomedical interest. The goal of this study was to design, synthesize, and characterize a novel thrombin-activated near-infrared fluorescence (NIRF) probe. The probe consisted of a thrombin-cleavable peptide spacer (...D-Phe-Pip-Arg...; Pip=pipecolic acid) and contained a terminal fluorescence reporter which was quenched when conjugated to a biocompatible delivery vehicle. A control peptide spacer was synthesized that differed by one amino acid. Following thrombin addition, the probe was activated within minutes. The NIRF signal increased by a factor of 27-fold within 20 minutes, and was inhibited by hirudin, a specific thrombin inhibitor. NIRF optical imaging experiments confirmed rapid activation of the probe in both buffer and human blood. The control probe showed minimal activation in all experiments. In addition to potentially furthering our understanding of thrombin regulation in vivo, the thrombin-activated near-infrared probe may have broad clinical application to the diagnosis of arterial and venous thrombosis.