Oats (Avena sativa L.) have received significant attention for their positive and consistent health benefits when consumed as a whole grain food, attributed in part to mixed-linkage (1-3,1-4)-β-d-glucan (referred to as β-glucan). Unfortunately, the standard enzymatic method of measurement for oat β-glucan is costly and does not provide the high-throughput capability needed for plant breeding in which thousands of samples are measured over a short period of time. The objective of this research was to test a microenzymatic approach for high-throughput phenotyping of oat β-glucan. Fifty North American elite lines were chosen to span the range of possible values encountered in elite oats. Pearson and Spearman correlations (r) ranged from 0.81 to 0.86 between the two methods. Although the microenzymatic method did contain bias compared with the results for the standard streamlined method, this bias did not substantially decrease its ability to determine β-glucan content. In addition to a substantial decrease in cost, the microenzymatic approach took as little as 6% of the time compared with the streamlined method. Therefore, the microenzymatic method for β-glucan evaluation is an alternative method that can enhance high-throughput phenotyping in oat breeding programs.