SFC separation on packed column for lipids analysis is well adapted thanks to the interclass lipid species separation as normal phase liquid chromatography1. The characterization of the lipidome typically requires the use of Data Dependent Acquisition (DDA) MS/MS method, which is limited to the fragmentation to the most intense signals during a cycle time2. Considering this issue, the strategy involving the iterative ion exclusion consisted of automatically placing prior fragmented ions on a list along sequential repetitive injections seems well adapted3. However, some MS/MS spectra at lower intensity present fragment ions of interest with weak signal-to-noise ratio. In this context, MS/MS acquisition method parameters are critical to ensure a short cycle time that meets this minimum requirements for an optimal coverage of lipid species in complex biological matrices. SFC gradient and MS/MS parameters of the Q/TOF were optimized to address this issue. A mix of lipid standards representing of 27 lipid classes were injected to determine the optimized parameters for the highly confident structural elucidation of lipid species present in complex biological matrices