Background: The Coombs' test, which detects immunoglobulin or complement on RBC surfaces, has long been the standard for laboratory confirmation of immune-mediated hemolytic anemia (IMHA), a common cause of hemolytic anemia in the dog. This test, however, suffers from relatively low sensitivity. Optimization of test sensitivity would lead to fewer discrepancies between laboratory results and clinical diagnoses, and in some cases institution of appropriate therapy in a timely manner. Objectives: The objectives of this study were to 1) characterize the sensitivity and specificity of 2 canine Coombs' tests for the detection of IMHA, 2) document the efficacy of using multiple antisera dilutions beyond what is directed by manufacturers, and 3) evaluate the necessity of monovalent antisera in the test protocol. Methods: Sixty-five canine whole-blood samples submitted for Coombs' testing were evaluated. Patients were classified as IMHA positive or negative based on a set of predetermined criteria. IMHA classification was compared to Coombs' test results from 2 different Coombs' tests adapted to a microtiter-plate format. One Coombs' test (VMRD Coombs' test) utilized a single polyvalent antiserum (VMRD, Inc, Pullman, WA, USA), while a second Coombs' test (University of Minnesota [U of MN] Coombs' test) used both polyvalent and monovalent antisera. Results: Sensitivity and specificity were 61% and 100% for the VMRD Coombs' test, and 82% and 95% for the U of MN Coombs' test. The use of multiple antisera dilutions resulted in 6 additional Coombs' positive test results. All positive Coombs' test results were positive by polyvalent antisera. Conclusions: When used in a microtiter-plate format, the U of MN Coombs' test was a more sensitive test for the detection of IMHA in canine patients when compared to the VMRD Coombs' test. The use of multiple antisera dilutions increased test sensitivity. Sensitivity, however, was not increased by the use of monovalent antisera in the Coombs' test protocol.