Gluten proteins play a key role in the unique baking quality of wheat by determining the water absorption capacity, cohesivity, viscosity, and elasticity of the dough. However, gluten from wheat, barley, rye, and oat can induce gluten sensitivity as well as celiac disease in susceptible populations. Hence, the gluten levels in foods labeled “gluten free” should be monitored. In this study, gluten-containing samples (sample: 600 g, water: 390 g) were treated with the commercial enzyme Protamex® (0.1-0.3% of sample weight) for 1-4 h and then measured with three ELISA kits. In the more viscous sample after treatment with 0.1% Protamex® for 1 h, the measured gluten contents were 1,802.6, 1,718.6, and 1,698.7 mg/kg using the G12, GLUTEN-CHECK, and Wheat/gluten (Gliadin) ELISA kits, respectively. The sample treated with 0.3% enzyme for 4 h had a lower viscosity (32.2 cps), and all three kits gave its gluten content as around 8.4 mg/g. When gluten breaks down, it does not act as gluten and its degradation is due to the enzyme. However, even when Protamex® was used at the same concentration for the same time, the measured values seem to be different for samples with and without the final heating treatment.