Many physiological processes function efficiently within a well-controlled pH range. Higher acidity level has been implicated with a number of systemic pathologies. The potential of pH sensitive fluorescent probes for reporting on biological environments has been widely utilized in a variety of cell studies and has been recently recognized as a powerful technique for in vivo imaging of diseases associated with elevated acidity level. We present several strategies for the development of pH sensitive probes suitable for in vivo imaging. The strategies include incorporation of pH sensitive functionalities in known fluorophores, synthesis of novel pH sensitive skeletons, and design of pH sensitive nanoparticles using acid-degradable polymers.