Isotropic bicelles are a widely used membrane mimetic for structural studies of membrane proteins and their transmembrane domains. Simple and cheap in preparation, they contain a patch of lipid bilayer that reproduces the native environment of membrane proteins. Despite the obvious power of bicelles in reproducing the various kinds of environments, the vast majority of structural studies employ the single lipid/detergent system. On the other hand, even if the alternative bicelle composition is used, the properties of mixtures are not characterized, and the mere presence of lipid bilayer and discoidal shape of bicelle particles is not confirmed. Here we present an extensive investigation of various bicellar mixtures and describe the behavior of bicelles with lipids other than classical DMPC, namely sphingomyelins (SM), phosphatidylethanolamines (PE), phosphatidylglycerols (PG), phosphatidylserines (PS), and cholesterol. These lipids are rarely used in modern structural biology, but can help a lot in understanding the influence of the membrane composition on the properties of both integral and peripheral membrane proteins. Additionally, the ability of diheptanoylphosphatidylcholine (DH7PC) to serve as a rim-forming agent was investigated. We followed the phase transitions as revealed by31P NMR and size of particles measured by1H NMR diffusion as the criteria of the proper morphology and structure of bicelles. As an outcome, we state that SM exclusively, and PG/PS in mixtures with zwitterionic lipids can form small isotropic bicelles, which reproduce the key features of lipid behavior in bilayers. Mixtures, containing exclusively the anionic lipids, fail to reveal the lipid phase transition and do not follow the size predicted for the ideal bicelle particles. PE and DH7PC are the unwanted components of bicellar mixtures, and cholesterol can be added to bicelles, however, with certain precautions. In combination with our several most recent works, this study provides a practical guide for the preparation of small isotropic bicelles.