Background: Since its introduction to the analytical community, the R5 method to quantify gluten led to a strong improvement of the situation for the food industry and celiac patients. During recent years, some questions arose on the use of the Codex Alimentarius factor of two to convert from prolamins to gluten, an overestimation of rye and barley, inadequate detection of glutelins, and the inhomogeneous distribution of gluten in oats. These limitations of the R5 method, especially when measuring oat samples, led to AOAC Standard Method Performance Requirement (SMPR®) 2017.021, which was approved by stakeholders in 2017. Objective: We present a collaborative study of a method for the quantitative analysis of wheat, rye, and barley gluten in oat and oat products using a sandwich ELISA that is based on four different monoclonal antibodies including the R5 monoclonal anitbody. Methods: The sandwich ELISA detects intact gliadins and related prolamins from rye and barley, high-molecular-weight (HMW) glutenin subunits (GS) from wheat, HMW secalins from rye, and low-molecular-weight (LMW) GS from wheat. It does not detect D-hordeins from barley. Samples are extracted by Cocktail solution, subsequently followed by 80% ethanol, and analyzed within 50 min. Results: The measurement range is between 5 and 80 mg/kg gluten using a calibrator made out of a gluten extract from four different wheat cultivars. The results of the collaborative test with 19 participating laboratories showed recoveries ranging from 99 to 137% for all three grain sources. Relative reproducibility SDs for samples >10 mg/kg gluten ranged from 10 to 53%. Conclusions: The collaborative study results confirmed that the method is accurate and suitable to measure gluten from all three grain sources and has demonstrated performance on oat matrices, which meets the criteria as specified in SMPR 2017.021. Data from in-house validation experiments are available as Annex B to this publication.