Background: The quantification of plasma glucagon and oxyntomodulin is important in the assessment of α-cell function, which is impaired in patients with diabetes. We aimed to transfer between laboratories a novel assay that uses liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the sensitive and specific measurement of these highly homologous hormones. Methods: Simultaneous measurement of glucagon and oxyntomodulin used immunoaffinity enrichment and LC-MS/MS. Immunoenrichment used free-for-research monoclonal antibodies (deposited at the Iowa Hybridoma Bank). Calibration of glucagon was achieved with a pure standard (characterized for purity and concentration, available to others). A detailed standard operating procedure was shared between three laboratories and the performance of the method was evaluated with samples collected from fasting patients with and without diabetes. Method comparison was made with two commercially available, FDA-registered glucagon immunoassays. Results: The method was linear at all three sites over the normal range (1-20 pM). When measured in duplicate, the median interlaboratory imprecision (%CV) of the measurement of 40 samples was 6.3% (IQR 4.5%) and 14.4% (IQR 12.4%) for glucagon and oxyntomodulin, respectively. Method comparison with commercially available immunoassays demonstrated good (Mercodia, R=0.92) or fair (Ansh, R=0.70) agreement. Multivariable linear regression using LC-MS/MS glucagon and oxyntomodulin concentrations to predict immunoassay results indicated significant cross-reactivity of each immunoassay with oxyntomodulin. Conclusion: We have validated a sensitive and specific assay for glucagon and oxyntomodulin that can be deployed in high-complexity clinical laboratories for research or the care of patients. Commercially available glucagon immunoassays have significant interference from molecules other than glucagon.