In vitro starch digestibility assays are more reproducible and less expensive than in vivo assays and are therefore important tools which assist breeding cereal cultivars with desirable starch digestibility. However, current in vitro starch digestibility assays often use non-mammalian digestive enzymes combined with the glucose oxidase peroxidase colorimetric (GOC) method of glucose detection which requires multiple steps. A new, simple, in vitro rice starch digestibility assay is developed by trialling several combinations of mammalian digestive enzymes and comparing blood glucose meter based glucose detection methods with the classic GOC method. The glucometer based detection methods had wider glucose concentration detection windows with good reproducibility compared with the GOC method. Digestion with a rat intestinal acetone powder (RIAP) alone was comparable (R2 > 0.99) with digestion using a combination of α-amylase, pepsin, pancreatin, and RIAP. The optimized in vitro starch digestibility assay correctly estimated the digestibility of rice samples which differed by in vivo glycemic index (GI). This rapid accurate assay only requires a small quantity of sample (15 mg) and can meet the high-throughput phenotyping requirement of breeding programs in order to develop healthy cereal grains such as low GI rice.