Ion mobility measurements of productions were used to characterize the collisional cross section (CCS) of various complex lipid [M-H]− ions using traveling wave ion mobility mass spectrometry (TWIMS). TWIMS analysis of various product ions derived after collisional activation of mono- and dihydroxy arachidonate metabolites was found to be more complex than the analysis of intact molecular ions and provided some insight into molecular mechanisms involved in product ion formation. The CCS observed for the molecular ion [M-H]− and certain product ions were consistent with a folded ion structure, the latter predicted by the proposed mechanisms of product ion formation. Unexpectedly, product ions from [M-H-H2O-CO2] − and [M-H-H2O]− displayed complex ion mobility profiles suggesting multiple mechanisms of ion formation. The [M-H-H2O]− ion from LTB4 was studied in more detail using both nitrogen and helium as the drift gas in the ion mobility cell. One population of [M-H-H2O]− product ions from LTB4 was consistent with formation of covalent ring structures, while the ions displaying a higher CCS were consistent with a more open-chain structure. Using molecular dynamics and theoretical CCS calculations, energy minimized structures of those product ions with the open-chain structures were found to have a higher CCS than a folded molecular ion structure. The measurement of product ion mobility can be an additional and unique signature of eicosanoids measured by LC-MS/MS techniques.