Polyphosphate (polyP) is a linear polymer of a few to many hundreds of phosphate (Pi) residues linked by high-energy phosphoanhydride bonds (Figure 1A). This ubiquitous polymer is found in bacteria, protists, and mammalian cells, and it was likely present prebiotically [1]. In bacteria, polyP accumulates in volutin or metachromatic granules, which are equivalent to acidocalcisomes [2]. In eukaryotic cells, polyP is present in different compartments, including the cytosol, nucleus, lysosomes, and mitochondria, but is preferentially accumulated in acidic vacuoles such as the yeast vacuole and acidocalcisomes [1], [3]. In these organelles, polyP, which is negatively charged, is in close association of inorganic (Ca2+, Mg2+, Zn2+, Fe2+, Na+, K+) and organic (basic amino acids, polyamines) cations. PolyP also combines with calcium and polyhydroxybutyrate forming channels in bacterial membranes, which make them competent for DNA entry; in mitochondria, as part of the mitochondrial permeability transition pore; and in the plasma membrane, as part of the potassium channel and the calcium pump (reviewed in [1], [4]). PolyP is arbitrarily divided into two forms: short-chain (from 3 to ∼300 Pi) and long-chain (from 300 to ∼1000 Pi) polyP, based on the method used for its extraction. For the detection of polyP, several methods have been described and a few examples are shown in Figure 1B–F. Figure 1 Methods used to detect polyP in cells.