Lipid nanodiscs provide a native-like lipid environment for membrane proteins, and they have become a valuable platform for the study of membrane biophysics. A range of biophysical and biochemical analyses are enabled when membrane proteins are captured in lipid nanodiscs. Two parameters that can be controlled when capturing membrane proteins in lipid nanodiscs are the radius, and hence the surface area of the lipid surface, and the composition of the lipid bilayer. Despite their emergence as a versatile tool, most studies with lipid nanodiscs in the literature have focused on nanodiscs of a single radius with a single lipid. In light of the complexity of biological membranes, it is likely that nanodiscs with multiple membrane components would be more sophisticated models for membrane research. It is possible to prepare nanodiscs with more complex lipid mixtures to probe the effects of lipid composition on several aspects of membrane biochemistry. Detailed protocols are described here for the preparation of nanodiscs with mixtures of phospholipids, incorporation of cholesterol, and incorporation of a spectroscopic lipid probe. These protocols provide starting points for the construction of nanodiscs with more physiological membrane compositions or with useful biophysical probes. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Assembly of mixed lipid nanodiscs Basic Protocol 2: Assembly of nanodiscs with cholesterol Basic Protocol 3: Incorporation of laurdan into nanodiscs for membrane fluidity measurements.