pH homeostasis is strictly controlled at a subcellular level. A deregulation of the intra/extra/subcellular pH environment is associated with a number of diseases and as such, the monitoring of the pH state of cells and tissues is a valuable diagnostic tool. To date, only a few tools have been developed to measure the pH in living cells with the spatial resolution needed for intracellular imaging. Among the techniques available, only optical imaging offers enough resolution and biocompatibility to be proposed for subcellular pH monitoring. We present herein a ratiometric probe based on upconversion nanoparticles modified with a pH sensitive moiety for the quantitative imaging of pH at the subcellular level in living cells. This system provides the properties required for live cell quantitative imaging i.e. positive cellular uptake, biocompatibility, long wavelength excitation, sensitive response to pH within a biologically relevant range, and self-referenced signal.