This chapter provides the optical measurement of the ion concentrations and the transmembrane potential of lysosomes. There are vast possibilities to measure ion concentrations using fluorescent ion sensors. The low pH is important for the activity of lysosomal enzymes and for various membrane trafficking steps. The electrogenic proton pumping requires a parallel electrical shunt to prevent a rapid build up of an inside-positive potential that would inhibit further pumping by the V-ATPase. This could be the influx of anions such as chloride (Cl-) and/or efflux of cations such as sodium (Na+) or potassium (K+). Another ion that has gained much attention for its importance in lysosomal function is calcium (Ca2+), whose luminal concentration may be up to 10,000-fold higher inside these organelles than in the cytosol. In addition to these ions, many further inorganic ions— such as zinc, iron, phosphate, magnesium, and copper— can be found in lysosomes and lysosomes play an important role in their cellular homeostasis.