Propidium iodide (PI) belongs to a class of intercalating nucleic acid probes; this family of compounds includes other popular fluorophores such as acridine orange. Unlike acridine orange, however, PI contains two positive charges, which confers it high affinity for the negatively charged DNA in cells, but also prevents it from passing through the hydrophobic lipid bilayer. Because of this characteristic, propidium iodide is often used to quantify cell viability, by differentiating healthy cells, where it is excluded, from dead cells, whose compromised membranes allow staining.

Cell viability assessments in flow cytometry often make use of propidium iodide alongside acridine orange. These two probes are chosen because they are well excited by the ubiquitous 488 nm argon ion laser. In these assays, propidium iodide is used to identify dead cell populations while acridine orange, which is cell permeable, quantifies healthy cells.

Another flow cytometry application is the usage of propidium iodide in differentiating apoptosis and necrosis. In these applications, PI is paired with an Annexin V conjugate, such as Annexin V-FITC, wherein the former stains the necrotic cells, with compromised membranes, while the latter stains the apoptotic cells, by binding to translocated phosphatidylserine.

Propidium iodide can be dissolved in several different solvents, both aqueous and organic, such as ethanol, DMSO, dimethyl formamide, PBS and water. The table below includes each solvent's respective solubility: