A reagent conservative Dot-enzyme immunoassay (Dot-EIA) was developed primarily for canine babesiosis caused by Babesia canis. The technique is simple, specific, and sensitive. All steps were carried out at room temperature. Strong agreement was observed between Dot-EIA and the conventionally used indirect immunofluorescence test. The procedure is adaptable to other protozoal disease, e.g., bovine babesiosis and human malaria.