Diffuse optical imaging (DOI) takes advantage of the relative ‘transparency’ of biological tissue in the near infrared that allows light to penetrate fairly deeply (several centimeters). The primary tissue contrast used by DOI is given by hemoglobin content, affording measurements of blood volume/angiogenesis and tissue oxygenation. DOI instrumentation is fairly compact and can be easily transported for bedside use, and the use of optical fibers for light delivery and collection allows easy integration of optical measurements with traditional medical imaging methods. DOI is still in the research phase, but significant clinical translation work has been done in breast cancer diagnosis and therapy monitoring, as well cerebral health assessment. Blood volume contrast has been shown to differentiate benign from malignant breast lesions, as well as predict chemotherapy outcome early in the course of treatment. At the same time, cerebral oxygenation and the cerebral metabolic rate of oxygen have been shown to correlate with perinatal brain injury. The main challenges on the path to clinical adoption are the need to demonstrate effectiveness in large-scale clinical trials and the need to develop targeted near-infrared contrast agents that can offer additional disease information beyond blood volume and hemoglobin oxygenation.