Introduction Glucagon and oxyntomodulin are peptide hormones differentially released from proglucagon that function in regulating blood glucose concentrations. Their overlapping amino acid sequences make the development of specific immunoassays difficult, but the specificity of liquid chromatography-tandem mass spectrometry can be used to distinguish the peptides. We aimed to develop a sensitive and specific mass spectrometric assay that uses non-proprietary reagents and normal-flow liquid chromatography in the simultaneous quantification of both analytes. Methods Bulk plasma proteins were precipitated in ethanol/ammonium hydroxide. Analytes were enriched using monoclonal antibodies generated in-house and analyzed using liquid chromatography-tandem mass spectrometry. A purified primary glucagon calibration material was sourced commercially and characterized for purity and concentration by HPLC-UV and amino acid analysis. Single point calibration was used to minimize between-day variability. Results The novel antibodies performed acceptably (peptide recovery 45-59%). The assay was precise (<13 %CV) and linear over the range of 1.3-14.7 pM and 1.1-13.7 pM for glucagon and oxyntomodulin, respectively. The primary calibrator concentration was determined to be 1.596 mg/g. Tube-type studies supported the use of protease inhibitor tubes at the time of blood draw. Patients with type 1 diabetes had lower concentrations of plasma glucagon when maintained on an insulin pump, but not with injectable insulin. Conclusion We have validated a method with a highly detailed standard operating procedure. We have characterized a reference material to help maintain accuracy and achieve between-assay and between-laboratory harmonization. The assay will be beneficial in better understanding α-cell health and glycemic control in diabetes and other diseases.