For more than 20 years, anthelmintics of the macrocyclic lactone (ML) drug class have been widely and effectively used as preventives against the canine heartworm, Dirofilaria immitis. However, in recent years an increased number of lack of efficacy (LOE) cases are being reported, in which dogs develop mature heartworm infections despite receiving monthly prophylactic doses of ML drugs. While this situation is raising concerns that heartworms may be developing resistance to MLs, compelling evidence for this is still lacking. Resolution of this dilemma requires validated biological or molecular diagnostic assays, but, unfortunately, no such tests currently exist. To address this need, we developed and optimized a larval migration inhibition assay (LMIA) for use with D. immitis third-stage larvae. The LMIA was used to measure the in vitro dose–response of two ML drugs (ivermectin and eprinomectin) on a known ML-susceptible laboratory strain of D. immitis. A nonlinear regression model was fit to the dose–response data, from which IC50 values were calculated; the mean IC50 and 95% confidence interval for IVM was 4.56 μM (1.26–16.4 μM), greater than that for EPR at 2.02 μM (1.68–2.42 μM), and this difference was significant (p = 0.0428). The R2 value for EPR assays (0.90) was also greater than that for IVM treatment (0.71). The consistency and reproducibility of the dose–response data obtained with this assay suggests that it may be a useful technique for investigating the relative susceptibilities to ML drugs in other D. immitis populations.