With the Global Program for the Elimination of Lymphatic Filariasis continuing to make strides towards disease eradication, many locations endemic for the causative parasites of lymphatic filariasis are realizing a substantial decrease in levels of infection and rates of disease transmission. However, with measures of disease continuing to decline, the need for time-saving and economical molecular diagnostic assays capable of detecting low levels of parasite presence is increasing. This need is greatest in locations co-endemic for both Wuchereria bancrofti and Brugia parasites because testing for both causative agents individually results in significant increases in labor and reagent costs. Here we describe a multiplex, TaqMan-based, real-time PCR assay capable of simultaneously detecting W. bancrofti and B. malayi DNA extracted from human bloodspots or vector mosquito pools. With comparable sensitivity to established singleplex assays, this assay provides significant cost and labor savings for disease monitoring efforts in co-endemic locations.