The location of double bonds within unsaturated lipids can greatly affect their biological functions. Liquid chromatography coupled to mass spectrometry (LC/MS) has become a powerful tool for lipid separation and structural elucidation. However, the determination of double bond position remains challenging for both conventional MS and tandem MS (MS/MS) due to the lack of product ions arising from fragmentation at the double bonds. Ozone can specifically react with carbon-carbon double bonds, which generates ozonolysis products with predictable masses that could be used for the assignment of double bond positions. In this work, ozonolysis reaction coupled in-line with mass spectrometry (in-line O3-MS) was developed for the unambiguous determination of double bond positions in unsaturated lipids. The in-line O3 device was composed of a gas-permeable, liquid-impermeable Teflon tube passing through a glass chamber filled with the ozone gas. Unsaturated lipids in the mobile phase of LC passed through the semi-permeable tube where they rapidly reacted with the ozone that penetrated through the tubing wall. The ozonolysis products carried by the LC mobile phase were then detected by MS in real-time. The in-line O3-MS method was successfully applied to mono- and poly-unsaturated fatty acid methyl ester (FAME), in which ozonolysis product aldehydes from the oxidative cleavage of each double bond were detected as protonated molecular ions under atmospheric pressure photo ionization (APPI) in positive ionization mode and used for the double bond localization. The in-line O3-MS is compatible with LC separations which are required in the analyses of complex lipid mixtures. Silver ion LC coupled to O3-MS (Ag+-LC/O3-MS) was used for the identification of conjugated linoleic acid (CLA) isomers from various sources. The diagnostic ions of the aldehydes resulting from the ozonolysis of the conjugated double bonds were used to identify the positional isomers, while at the same time the geometry of the double bond (cis or trans) could be determined by the elution order in Ag+-LC. The O3-MS method was also expanded to double bond localization in phospholipids (PL). For complex PL extract, heart-cut two dimensional LC (2D-LC) using hydrophilic interaction liquid chromatography (HILIC) for PL class separation and C18 reverse phase LC for molecular species separation was achieved using a 10-port 2-position switching valve (HILIC×C18 LC). The PL species were firstly identified using electrospray ionization (ESI) in negative ionization mode with MS/MS analysis applied for the composition determination of the fatty acyl chains on the glycerol backbone. The ozonolysis device was then placed in-line between the 2D-LC and ESI source (HILIC×C18 LC/O3-MS), and the ozonolysis product aldehydes allowed unambiguous assignment of double bond positions in both fatty acyl chains of PL. Compared to the previous methods used for double bond localization in lipids, no off-line derivatizations are needed and also the O3-MS spectra are much simpler for interpretation. The in-line ozonolysis experiment can be easily incorporated into many existing LC/MS methods, and applied complementary with current lipidomic analysis. In conclusion, the LC/O3-MS method can provide insight into the specific structure of lipid isomers and further reveal the complexity of the lipidome.