Fluridone has been used to successfully manage Eurasian watermilfoil since the late 1980s. However, recent documentation of hybrid watermilfoils and the resulting potential for reduced herbicide sensitivity necessitate the need for an assay to determine individual population response to fluridone. A known fluridone-resistant hybrid watermilfoil population from Townline Lake in Michigan was compared to 11 Eurasian and hybrid watermilfoil populations in laboratory experiments to develop a method for determining response to fluridone. Apical shoot tips were exposed to increasing concentrations of fluridone (0 to 48 μg L−1) for 3, 5, and 7 d. Chlorophyll fluorescence (Fv/Fm) was evaluated using a pulse-amplitude modulated fluorometer at each interval along with pigment analysis of chlorophyll and β-carotene at the 7-d interval. Fv/Fm and pigment analysis yielded the same results. A fluridone concentration of 12 μg L−1 and an analysis interval of 7 d were found to be optimal in determining invasive watermilfoil response to fluridone. Use of such small-scale assays can provide resource managers a rapid tool to cost-effectively evaluate invasive watermilfoil response to fluridone.