The maintenance of pH homeostasis is critical for a variety of cellular metabolic processes. Although ocean acidification is likely to influence cellular metabolism and energy balance, the degree to which intracellular pH in phytoplankton differs from the external environment under varying environmental pH levels is not well characterized. While there are numerous existing methods for the determination of intracellular pH in the form of single peak emission (e.g., BCECF) and radioisotopic (e.g., 14C-DMO) indicators for use with phytoplankton, the fluorescent pH indicator seminaphtharhodafluor (SNARF) has not been established as a robust method for measuring in vivo pH in phytoplankton. SNARF has superior accuracy and sensitivity since it exhibits dual emission peaks from a single excitation wavelength and the ratio of the two are related to pH. The use of a ratio limiting variations in fluorescence due to dye loading, photobleaching, and instrument variation; moreover, like other fluorescence-based assays, it does not require the specialized equipment and permits that radioisotopic methods do. As a first step, we tested the performance of SNARF for measuring intracellular pH in vivo in a number of phytoplankton taxa. SNARF detection was accomplished using fluorescence spectroscopy (FS) and laser scanning microscopy (LSM). Since SNARF fluorescence is activated by cleavage of an ester group from the core fluorophore by nonspecific esterases, we measured esterase activity using fluorescein diacetate (FDA) to characterize variability in esterase activity among phytoplankton taxa, with a view towards its influence on assay performance. Esterase activity cell volume; however, there was no indication that enzyme specificity and differences in individual esterase profiles adversely affected SNARF performance in phytoplankton. Assays of intracellular pH using SNARF were comparable to those made with 14C-labeled DMO, an accepted standard method. Thus, SNARF provides robust measurements of intracellular pH in phytoplankton, constituting a useful tool in investigations of the effects of ocean acidification and fluctuations in environmental pH on cellular physiology.