It is often difficult to continuously monitor and quantify inflammatory responses in vivo. These dynamic responses however are often accompanied by specific pH changes. A new ratiometric optical pH probe is developed by combining pH-sensitive (CypHer5E) and pH-insensitive (Oyster800) fluorescent dyes into nanoparticles for in vivo optical imaging. By taking the ratio of fluorescence intensities at different wavelengths, these nanosized sensors provide excellent measurement capabilities, and unique mapping, of the continuous in vivo pH changes for three different inflammation models. In each model a strong positive correlation is found between ratiometric pH changes and the corresponding inflammatory response measured by histological analyses. These results indicate that ratiometric imaging can provide a noninvasive, rapid, and highly sensitive optical readout for the pH-ratio changes in vivo. Furthermore this technique may be used to monitor the real-time dynamics of inflammatory processes.