Routine screening for heartworm disease in dogs, caused by Dirofilaria immitis, requires whole blood, serum, or plasma and can be performed using point-of-care antigen tests. If heat treatment of serum is necessary or repeat testing is performed, veterinarians may not have the adequate serum volume necessary to complete the in-house antigen testing. To avoid repeated serum collection from patients, we investigated whether diluting D. immitis-positive canine serum with ethylenediaminetetraacetic acid (EDTA) at a 1:1 ratio can increase sample volume without compromising antigen detection. Forty-eight confirmed D. immitis antigen-positive canine serum samples were diluted with EDTA and aliquoted into two subsets, one subset receiving heat treatment and one remaining non-heat-treated. Samples were then evaluated by eight commercially available heartworm antigen tests according to the manufacturer’s instructions. With the diluted serum, we found no significant difference in test sensitivities in either the heat-treated or non-heat-treated groups when compared to non-diluted serum results. These findings imply that with further investigation, it may be possible to add an EDTA dilution to heartworm antigen testing protocols when working with limited serum volume.