Non-ionic detergents are key reagents for the characterization of membrane protein drug targets. Suitable detergents are commonly identified by trial and error, which leads to failed preparations and raising costs. Recent findings suggest that not only the chemistry of detergents but also the strategy with which detergents and proteins are brought together impact the successes of investigations. To facilitate the future development of non-ionic detergents in membrane protein research, herein, we review chemical design concepts and detergent exchange strategies for a successful integration of detergents into the analysis of challenging membrane protein complexes. Our overview reveals exciting opportunities to tackle existing challenges, including the stabilization of G-protein coupled receptors, the development of fluorinated detergents for studying protein-lipid binding, top-down design of detergents, and hybrid detergents for the identification of non-canonical lipid associations to proteins with relevance to antibiotic research. Our review will facilitate the development of chemical tools for the biophysical characterization of membrane proteins and support the discovery of biological findings in the future.