The purpose of this study was to define the operational and performance characteristics of a commercially available monoclonal antibody based (mac) ELISA for detection of allergen-specific IgE in dogs. The average intra-assay variance over 1 year was 9.7% (range 2.5–62.7%), while the interassay variance averaged 10.8% (range 8.1–13.8%). The average positive control responses observed for grass, weed, tree and mite allergens during each month remained relatively constant; the average monthly variance was 11.6% (range 8.3–19.2%) for grass pollens, 13.3% (range 9.1–20.4%) for weed pollens, 13.3% (range 9.8–18.2%) for tree pollens and 13.6% (range 8.9–18.7%) for mite allergens. The interlaboratory concordance of results for the macELISA was approximately 91%. The interlaboratory concordance of results comparing the macELISA and a high affinity IgE receptor-based ELISA was approximately 92%. The results demonstrate that the macELISA is reproducible and the results are comparable to the high affinity IgE receptor based ELISA within and between laboratories.