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Size exclusion chromatography (SEC) in combination with optical measurements has become a popular form of analysis to characterize dissolved organic matter (DOM) as a function of molecular size. Here, SEC coupled with in-line absorbance scans and fluorescence emission scans was utilized to derive apparent fluorescence quantum yield (Φf) as a function of molecular weight (MW) for DOM. Individual instrument-specific SEC-fluorescence detector correction factors were developed by comparison of an SEC-based excitation emission matrix (EEM) to an EEM generated by a calibrated benchtop fluorometer. The method was then applied to several sample sets to demonstrate how to measure the Φf of unknown DOM samples and to observe changes to Φf following a processing mechanism (ozonation). The Φf of riverine water samples and DOM fulvic acid isolates from Suwannee River and Pony Lake increased from < 0.5% to a maximum of ∼2.5–3% across the medium- to low-MW range. Following ozonation of PLFA, Φf increased most notably in the large-MW fractions (elution volumes < 40 mL). Overall, this method provides a means by which highly fluorescent size fractions of DOM can be identified for more detailed analyses of chemical composition and its changes through different processing mechanisms.