Criegee intermediates (CIs) play a significant role in cell membrane peroxidation, but their identification remains elusive at the molecular level. Herein, we combined interfacial extraction and sonic spray ionization mass spectrometry to study the oxidation reaction of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) mediated by ozone (O3) at/near the surface of a hung water droplet. On-line interfacial extraction and ionization provided a snapshot of the short-lived CIs. Experiments in which the content of water was varied provided evidence for the formation of CIs, which has not been previously observed. Capture experiments using 5,5-dimethyl-pyrroline N-oxide (DMPO) indicated that CIs could be selectively characterized, and the extracted ion current (EICs) of CIs vs DMPO-CI adducts further confirmed the successful observation of CIs. Theoretical calculation suggested that surface ozonolysis of POPG was mainly mediated by anti-CI. These results open a new route for aqueous surface reactive species identification, and benefit toward the understanding of disease development associated with cell oxidative stress mediated by CIs.