Inflammation is associated with many diseases, such as stroke, cancer, and atherosclerosis. Noninvasive in vivo monitoring of inflammation can provide deeper understanding of such diseases, which might help to develop better treatment. Inflammation normally causes neutrophils and macrophages to generate reactive oxygen species (ROS) as the destruction tool, which can be used as a biomarker for inflammation. Near- infrared (NIR) window is optimal for in vivo fluorescence imaging owing to the reduced autofluorescence and low attenuation of light in biological tissues. Among NIR fluorescent probes, activatable probes have the promise of achieving high imaging contrast. In this chapter, we describe the method for in vivo fluorescence imaging of inflammation using a ROS-activatable NIR probe.