Iron influences the climate system by limiting primary productivity. It is therefore essential to accurately measure the iron fraction associated with phytoplankton in aquatic systems. A washing procedure using EDTA, being efficient for numerous trace metals, is not strong enough to remove iron adsorbed to the surface of microorganisms. Stronger washing solutions are used for iron, but these have only been assessed for a marine diatom. This study assesses the applicability of the oxalate washing procedure for both fresh- and seawater aquatic systems. We assessed iron solubilization as a result of oxalate washing in both synthetic and natural freshwater and seawater, and we tested it on several model phytoplankton and natural assemblages from Lake Champlain, the Southern Ocean, and the Derwent River estuary. We report the effects of the oxalate solution contact time, concentration, and amendment. Our study shows that 20-min washing provides an efficient measurement of the intracellular phytoplanktonic pool of iron in both freshwater and seawater. The direct amendment of oxalate in the experimental solution presents many advantages that are critical for the measurement of size-fractionated particulate iron. These include fine control of bioaccumulation termination, a significant gain in time, and homogeneity of the washing treatment.