A protocol to assess the potential release of dissolved reactive phosphorus (DRP) by enzymatic hydrolysis of dissolved organic phosphorus (DOP) in waters (sediment porewater and sewage liquors in this study) under environmental conditions is presented. This protocol enables the quantification of different classes of DOP compounds using a variety of phosphatase enzymes, i.e., alkaline phosphatase, phosphodiesterase, and phytase. All experiments were carried out within the pH range of most natural waters, i.e., at neutral (pH 7) or slightly alkaline pH (pH 9). Tri-sodium citrate and sodium dodecyl sulfate (SDS) were used in the assays to prevent interferences due to adsorption processes in the presence of multivalent metallic cations and to minimize protein binding. Applying this protocol revealed that labile phosphate monoesters always represented the largest fraction of enzymatically hydrolyzed P in sewage liquors and sediment porewater. Total enzymatically hydrolyzable P (EHP) represented only 16% of the TDP in the sediment porewater but up to 43% in sewage liquors. Because most of the enzymes used in this study are likely to exist in aquatic ecosystems, the EHP fraction might represent a source of potentially bioavailable P of similar magnitude to DRP.