AbstractA targeted method for the quantification of bioavailable amide N found in marine DON (bDON) is presented. The method utilizes mild acid hydrolysis to convert amide N found in proteins and N‐acetyl amino polysaccharides to primary amine containing products that are measured using a highly sensitive (nanomolar range and precision) fluorometric technique with addition of O‐phthaldialdehyde. We find amidic bDON concentrations ranging from 0.08 to 1.82 μM N within waters from the upper 300 m in the southern California Current, Southern California Bight, and subtropical North Pacific representing 15–33% of bulk DON concentrations. Bioassay experiments from the North Pacific revealed consumption of ~20% of the in situ bDON within 5 days. The method represents a simple and rapid tool for the quantification of bioavailable DON concentrations in seawater with improved analytical precision over traditional estimates of bulk DON concentrations.