Dictyostelium amoebae are frequently used to study the organization and function of the endocytic pathway, and specific protocols are essential to measure the dynamics of endocytic compartments and their internal pH.We have revisited these classical protocols to measure more accurately endosomal pH, making use of a fluorescent probe (Oregon green) more adequate for very acidic pH values. This pH-sensitive probe was combined with a pH-insensitive marker, in order to visualize simultaneously endosome dynamics and pH changes. Finally, a flow cytometer was used to measure endosomal pH in individual cells.Using these simple protocols the endosomal pH of endocytic compartments can be assessed accurately, revealing the extreme acidity of Dictyostelium lysosomes (pH <3.5).