<p>An extremely sensitive amylase activity assay was developed using the natural substrate starch and two ancillary enzymes: a glucose oxidase (GOD) and a peroxidase, to measure the residual activity of the <i>α</i>-amylase from <i>Bacillus subtilis</i> in white bread. Firstly, the concentrations of the assay components: electron acceptor DA-67 (50&#xa0;μM), horseradish peroxidase (681 nkat mL⁻¹), a GOD from <i>Aspergillus niger</i> (1550 nkat mL⁻¹) and the natural substrate starch (0.01% (w/v)), were optimized to achieve high sensitivity. The linearity of the assay was then tested with both an endo- (<i>α</i>-amylase from <i>B. subtilis</i>) and exo-acting amylase (maltogenic amylase from <i>Geobacillus stearothermophilus</i>), and the effect of the incubation time on the assay sensitivity was investigated and optimized. The optimized assay was capable of determining a minimal amylase activity of 0.33 pkat mL⁻¹ for both amylases tested with an assay run time of 7.5&#xa0;h. This new DA-67 amylase assay demonstrated 4.7- and 4.2-times higher sensitivity, respectively, compared to optimized versions of the commercial Ceralpha (determination of endo-amylase activities) and Betamyl3 (determination of exo-amylase activities) assays. The new DA-67 amylase assay was used to determine the residual activity of <i>α</i>-amylase from <i>B. subtilis</i> in white bread. A consistent residual activity of 2.26 ± 0.15% was reliably determined.&#xa0;</p>