The use of phospholipid vesicles as reaction containers, as vehicles for pharmaceutical drug delivery, and as model systems for cells has prompted the development of new methods for analyzing the structure of vesicles and their contents. The pH of the interior of vesicles is of particular interest when analytes are encapsulated and concentrated with the use of a pH gradient. While the interior pH is generally measured for large populations of vesicles, we report the measurement of the interior pH of individual vesicles as their buffer contents are titrated by transfer of N-methylbutylamine (NMBA) into the vesicle by a pH gradient. The initially acidic buffer within the vesicles is titrated along with a small concentration of an encapsulated pH sensitive dye, 5,6-carboxy SNARF-1-dextran. Images of the indicator fluorescence from each vesicle and its dispersed fluorescence spectrum are recorded using epi-illumination spectral fluorescence microscopy. From a fit of the spectra to the respective acid and base forms of the fluorescent indicator, the interior pH of individual vesicles as a function of the concentration of the NMBA titrant in the external solution could be determined.