Centre de Recherche Clinique Etienne-Le Bel 1 article published in JoVE Biology Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy Fabrice Lucien1, Kelly Harper1, Pierre-Paul Pelletier1, Leonid Volkov2, Claire M. Dubois1 1Department of Pediatrics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, 2Biophotonics Core Facility, Centre de Recherche Clinique Etienne-Le Bel Several methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of cytoplasmic and organellar pH. Here, we describe in detail a rapid and accurate methodology to simultaneously measure cytoplasmic and vesicular pH by ratiometric imaging of living cells.