Blood-sucking insects are responsible for the transmission of several important disease-causing organisms such as viruses, bacteria, and protozoans. The hematophagous hemipteran Rhodnius prolixus is one of the most important vectors of Trypanosoma cruzi, the etiological agent of Chagas disease. Due to the medical importance of this insect, it has been used as a study model in physiology and biochemistry since the 1930s. Artificial feeding has been recognized as a feasible and a more ethical alternative method of feeding these hematophagous insects. To prevent clotting after blood collection defibrination or treatment with anticoagulants are necessary. Although anticoagulants have been routinely used for stabilizing the collected blood, there is a gap in demonstration of the effects of using anticoagulants on the feeding and development of the hematophagous insect Rhodnius prolixus. In this study, we compared the survival rate, molting efficiency, fertility, and infection development between insects that were fed on blood containing three different anticoagulants (citrate, EDTA, and heparin). We observed that fifth instar nymphs that were fed on blood containing EDTA and citrate could not perform digestion properly, which resulted in molting inefficiency. Adult insects that were fed on EDTA-containing blood laid lower number of eggs, and also had a diminished egg hatch percentage. When we delivered T. cruzi parasites in blood containing citrate or EDTA to the insects, a lower number of parasites and metacyclic trypomastigotes was observed in the intestine compared to the group fed on heparin-containing blood. Since heparin could potentially inhibit DNA polymerase activity in DNA samples extracted from the intestine, we analyzed different heparin concentrations to determine which one is the best for use as an anticoagulant. Concentrations ranging between 2.5 and 5 U/mL were able to inhibit coagulation without severely impairing DNA polymerase activity, thus indicating that this should be considered as the range of use for feeding experiments. Our results suggest that among the three anticoagulants tested, heparin can be recommended as the anticoagulant of choice for R. prolixus feeding experiments.