Measurements of pH in acidic cellular compartments of mammalian cells is important for our understanding of cell metabolism, and organelle acidification is an essential event in living cells especially in the endosomal-lysosomal pathway where pH is critical for cellular sorting of internalized material. Intracellular pH can be measured by the use of fluorescence ratio imaging microscopy (FRIM), however, available methods for pH measurements in living cells are not optimal. Nanoparticle based optical sensor technology for quantification of metabolites in living cells has been developed over the last two decades. However, even though these sensor systems have proven themselves as superior to conventional methods, there are still questions about the use of these sensors that need to be addressed, especially regarding sensor design and calibration. We have developed a new triple-labelled pH nanosensor designed with two pH sensitive dyes and one reference dye covalently attached to the nanoparticle matrix. The effective pH sensitivity range of this sensor was determined to be at least 3.1 pH units, which is twice the range of conventional dual-labelled nanosensors which is 1.4 pH units. The triple-labelled nanosensor was demonstrated to be superior to a dual-labelled nanosensor when performing measurements of pH in lysosomes in response to treatment of the cells with Bafilomycin A1. The triple-labelled nanosensor could follow the resulting increase in pH from a mean value around pH 4.3 up to 5.6, whereas the duallabelled nanosensor failed to measure the pH of up to 70% of the nanosensor containing vesicles. In order to perform reliable measurements of pH, proper calibration and image analysis have to be performed. We investigated nanosensors calibration and provide a suitable equation for fitting calibration curves which can be adapted to both dual- and triple-labelled sensors as well as sensors with even more sensitive dyes. Furthermore, we describe how image analysis can be performed correcting for both background fluorescence and differences in laser power. We further demonstrated the use of the triple-labelled pH nanosensor in answering biological questions. The triple-labelled nanosensor was shown to specifically localize in lysosomes where the pH was measured in response to the treatment of the cells with polyethylenimine (PEI), a potent transfection agent. We found no change in lysosomal pH within a timeframe of up to 24 h in response to any of the investigated PEIs. In relation to these findings we do not reject the “proton sponge” hypothesis, but suggest that the effect is not associated with changes in lysosomal pH. Finally, we investigated the pH profiles of a positively charged nanosensor in six different cell lines as well as the profile of a hyaluronic acid conjugated nanosensor tested in one cell line. After 24 h of incubations all sensors resided in compartments with low pH, recognized as lysosomes in HeLa cells, and responded with an increase in pH to the treatment with Bafilomycin A1. This indicates that all internalized material eventually ends up in the lysosomes, even though the hyaluronic acid conjugated nanosensor showed uptake directed by the CD44 receptor. The initial uptake pathway employed by this nanosensor could potentially be different from the one employed by the positively charged nanosensor. In conclusion, we have developed a triple-labelled pH nanosensor which was shown to be superior to conventional dual-labelled nanosensors with respect to the pH sensitive range. With proper calibration and image analysis we performed pH measurements of lysosomes in different mammalian cells in response to the transfection agent PEI and in relation to different functionalizations.