Although the mechanisms underlying gamete fusion during sexual reproduction remain obscure, recent studies have implicated the conserved transmembrane protein Hapless 2 (HAP2) as an ancestral gamete fusogen. In this regard, genetic disruption of the HAP2 locus in a variety of eukaryotic organisms, ranging from the ciliated protist Tetrahymena thermophila, to flowering plants and malaria parasites, has been shown to lead to a decisive block to fertilization due to the hapless cells’ inability to fuse their gametic membranes. To better study the role of HAP2 in membrane fusion we developed a flow cytometry-based assay for the quantification of cellular fusion during T. thermophila sexual conjugation. In this assay, complementary T. thermophila mating types are first labeled with fluorescent cell tracers (either carboxyfluorescein diacetate succinimidyl ester or cell trace far red) and then allowed to undergo mating. When membrane fusion occurs, the cells in a mating pair actively exchange a portion of their fluorescently-labeled cytoplasmic proteins. After mating, exconjugant cells disengage enabling easy quantification of the overall percentage of cells that exchanged cytoplasm along with the relative amount of cytoplasm transferred using flow cytometry. This assay allowed us to validate our previous finding that HAP2 is necessary for membrane fusion during T.thermophila fertilization and provided a rapid way to test for essential functional domains in the HAP2 protein using deletion analysis. We found that disruptions of core domains within the N’-terminus severely restricted membrane fusion events between mating cells, whereas deletions at the C’-terminus had little impact on fusogenicity. Importantly, through the use of homology modelling, we identified a region within the N’-terminal ectodomain that appears to have a high degree of structural similarity to the Dengue virus envelope protein (a class II viral fusogen). This region contains a peptide loop used by the viral protein to perturb target membrane bilayers enough to initiate fusion, allowing viral entry into host cells during infection. We found that deletion of either this ‘fusion loop’ or the entire Dengue envelope protein homology domain in T. thermophila HAP2 completely eliminated cytoplasmic exchange between mating Tetrahymena cells. In addition to raising interesting questions regarding the evolutionary origin of HAP2, these results may provide new insights into the mechanistic function of this key developmental protein.