Odd as it may seem, experimental challenges in lipid research are often hampered by the simplicity of the lipid structure. Since, as in protein research, mutants or overexpression of lipids are not realistic, a considerable amount of lipid research relies on the use of tagged lipid analogues. However, given the size of an average lipid molecule, special care is needed for the selection of probes, since if the size and intramolecular localization of the probe is not specifically taken into account, it may dramatically affect the properties of the lipids. The latter is particularly important in cell biological studies of lipid trafficking and sorting, where the probed lipid should resemble its natural counterpart as closely as possible. On the other hand, for biophysical applications, these considerations may be less critical. Here we provide a brief overview of the application of several lipid probes in cell biological and biophysical research, and critically analyze their validity in the various fields.