Drug-induced intracellular hydrogen peroxide overproduction is proposed as an upstream event in apoptosis signaling. It is therefore important to develop a sensitive method to quantify hydrogen peroxide in biological samples undergoing cell death. We report herein a luminol-based chemiluminescent assay with a suitable detection range of best linearity from 1 to 0.1 μM hydrogen peroxide with a CV range of 5.7% to 21.7% for intraday measurement at the detection range. We hypothesized that digitoxin-treated human hepatoma SK-Hep-1 cells produce hydrogen peroxide to cause cytotoxicity, which was verified by measuring the spent media by our chemiluminescent method in a dose-dependent manner. Taken together, our sensitive luminol-based chemiluminescent assay has potential use in the quantification of trace amounts of digitoxin overdose-induced hydrogen peroxide in biological samples.