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Abstract Aquatic organisms utilize dissolved organic phosphorus (DOP) when phosphate levels are insufficient to satisfy their P‐demand. Its critical role in supporting primary production has motivated efforts to characterize DOP composition and gain insight into its bioavailability.³¹P nuclear magnetic resonance (³¹P‐NMR) spectroscopy, the principal tool currently used by aquatic geochemists to identify compositional aspects of the bulk marine DOP pool, has provided limited compositional detail beyond the immediate P‐bonding environment, and does not provide quantitative information on potential bioavailability of DOP. As a result, the DOP pool remains poorly characterized, limiting predictive power relative to DOP bioavailability. We have developed a sequential ultrafiltration (SUF) method to segregate the DOP pool into four distinct molecular weight ranges. The concentrated molecular weight fractions are then incubated with phosphohydrolytic enzymes to determine the potential bioavailability of monoester‐ and diester‐bound P. DOP molecular weight segregation was verified using commercially available DOP compounds of known molecular weight as analogues for naturally occurring DOP. We have applied the SUF method to surface water samples from a nearshore transect in Kāne'ohe Bay, Hawai'i, and surface waters from oligotrophic Station ALOHA (Hawai'i Ocean Time Series). Results of these field tests indicate that the coupled SUF‐bioavailability method reveals greater compositional complexity of the marine DOP pool than has been resolved by³¹P‐NMR studies. Notably, the SUF‐bioavailability method does not require expensive, technically complicated instrumentation (e.g.,³¹P‐NMR), and thus is readily accessible to researchers wishing to probe marine DOP composition who do not have access to or expertise in³¹P‐NMR.