Lipid quantification on the molecular species level is based on the abundance of specific fragments detected in MSⁿ spectra of endogenous lipids and standards. As species-specific internal standards are commonly not feasible, lipids are quantified utilizing only one or few internal standard(s) per class. However, the analytical response of individual lipid species is determined by its lipid class and other structural elements like double bonds, hydroxy groups, and chain length. This tutorial covers developing and applying fragmentation models for sphingolipids and glycerophospholipids, aiming to overcome challenges in accurately quantifying these critical biomolecules. The determined response factors are based on experimental data and are independent of the employed instrumentation, collision energies, and internal standard(s).