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Cyanobacteria are the primary taxa responsible for freshwater harmful algal blooms (HABs), with several genera capable of producing potent intracellular toxins and off-flavor compounds. There is considerable growing interest in methods to rapidly quantify cyanobacteria in water samples. Past studies have demonstrated poor correlations between phycocyanin in vivo fluorescence and cyanobacterial cell densities. We conducted a series of laboratory experiments aimed at refining a protocol that uses benchtop fluorometry to measure the cyanobacterial pigment, phycocyanin, to accurately estimate cyanobacterial biovolume. In our study, we found strong correlations between phycocyanin concentration and cyanobacterial biovolume (but not for cell densities) both within and across ponds, which varied widely in productivity and algal diversity. Thus, benchtop fluorometry of phycocyanin is a viable method for water resource managers to quickly estimate cyanobacterial biovolume.