An 8-year-old Jack Russell Terrier was diagnosed with hemolytic anemia caused by hemoplasmosis 4 years following splenectomy. Quantitative real-time PCR (qPCR) analysis was used initially to confirm infection with Mycoplasma haemocanis and subsequently to monitor and direct medical therapy. Doxycycline was administered beyond resolution of clinical signs until hemoplasma DNA could no longer be detected by qPCR. The dog remained clinically healthy and hemoplasma-negative 8 months following cessation of therapy. Canine hemoplasmosis should remain as a differential diagnosis for hemolytic anemia in dogs, particularly those that are splenectomized or immunocompromised, even in geographic regions where prevalence of infection is low. Prolonged doxycycline administration has been shown by qPCR to lead to sustained absence of detectable infection and should be considered as a first line treatment for canine hemoplasmosis.