Non-invasive molecular imaging opens a window into the biology of living subject, and its success is critically dependent on the development of chemical probes to enable direct visualization of specific molecular targets and processes. This talk will highlight some of our efforts in building these molecules. The first example involves the design and synthesis of fluorogenic substrates for imaging the activity of bacterial enzyme beta-lactamase in mice to facilitate the detection and study of tuberculosis, one of the most frequent causes of death worldwide. In the second example, I will discuss the concept of developing biocompatible chemical reactions to synthesize nanostructures in cells and demonstrate its application for imaging of protease activity. These examples aim to underline the important role of chemistry in this rapidly evolving field of molecular imaging.