Unlike cytosolic proteins, membrane proteins (MPs) are embedded within the plasma membrane and the lipid bilayer of intracellular organelles. MPs serve in various cellular processes and account for over 65% of the current drug targets. The development of membrane scaffold proteins (MSP)-based nanodiscs has facilitated the accommodation of synthetic lipids to stabilize MPs. Moreover, bio-inspired synthetic polymers present an invaluable tool for the excision and liberation of superstructures of MPs and their surrounding annular lipid bilayer in the nanometric discoidal assemblies. The unprecedented range of membrane proteins (some with up to 24 transmembrane helices) from various sources have been reconstituted into nanodiscs, and both their conformational dynamics and interactions have been studied by cryo-electron microscopy (cryo-EM), solution-state, and solid-state nuclear magnetic resonance spectroscopy (NMR), X-ray crystallography, small-X-ray scattering (SAXS), and native mass spectrometry. In this collection, we will address how various biophysical approaches are utilized to characterize membrane protein assemblies and their interactome in nanodiscs.