To perform biochemical and structural studies of membrane proteins (MPs), amphiphilic molecules that mimic the hydrophobic environment of lipids are required. Over the past decades, detergents, a particular class of amphiphiles, have been the most widely used for MP study. However, detergents tend to be inactivating for MPs, which has prompted the recent design of alternative strategies. The present review focuses on fluorinated amphiphiles, also called fluorinated surfactants, whose hydrophobic tail is partially fluorinated. Fluorinated chains are lipophobic, bulkier, and more rigid than the hydrogenated ones. In consequence, fluorinated surfactants (F-surfactants) would poorly interfere with protein–protein, protein–lipid, and protein–cofactor interactions, thus contributing to the stability of solubilized MPs. Here, we first introduce the concepts motivating the exploration of different F-surfactant families. We then focus on the design and the surface and self-aggregation properties of two recent series—including some original compounds—of F-surfactants bearing branched polar heads. The promising biochemical applications of F-surfactants, from the literature from 2010, are reviewed. Finally, we provide an overview of other recently developed nonconventional amphiphiles with sugar-based head groups.