Measurement of intracellular pH can be readily accomplished using tools and methods described in this chapter. We present a discussion of technical considerations of various ratiometric pH-sensitive probes including dyes and genetically encoded sensors. These probes can be used to measure pH across physical scales from macroscopic whole-mount tissues down to organelles and subcellular domains. We describe protocols for loading pH-sensitive probes into single cells or tissues and discuss ratiometric image acquisition and analysis. © 2014 Elsevier Inc.