The ability to monitor hydrogen peroxide (H₂O₂) in aqueous smoke extracts will advance our understanding of the relationship between cigarette smoke-induced oxidative stress, inflammation, and disease and help elucidate the pathways by which the various smoke constituents exert their pathogenic effects. We have demonstrated, for the first time, the measurement of H₂O₂ production from cigarette smoke without prior separation of the sample. Cigarettes were tested on a commercial smoking machine, such that the whole smoke or gas vapor phase was bubbled through phosphate buffered saline solution at pH 7.4. Aliquots of these solutions were analyzed using an Amplex Red/horseradish peroxidase fluorimetric assay that required only a 2 minute incubation time, facilitating the rapid, facile collection of data. Catalase was used to demonstrate the selectivity and specificity of the assay for H₂O₂ in the complex smoke matrix. We measured ∼7–8 μM H₂O₂ from two reference cigarettes (i.e., 1R4F and 2R4F). We also observed 9× more H₂O₂ from whole smoke bubbled samples compared to the gas vapor phase, indicating that the major constituent(s) responsible for H₂O₂ formation reside in the particulate phase of cigarette smoke. Aqueous solutions of hydroquinone and catechol, both of which are particulate phase constituents of cigarette smoke, generated no H₂O₂ even though they are free radical precursors involved in the production of reactive oxygen species in the smoke matrix.