The plasma membrane is a ∼5nm lipid bilayer that ensheathes all living cells. Lateral heterogeneity and transbilayer asymmetry in lipid and protein distributions generate local variations in lipid packing, polarity, viscosity, lateral tension and curvature that regulate membrane protein conformation, nanoscale organisation and signalling. The composition and organisation are therefore tightly controlled and intimately coupled to cellular function. Thus, in parallel, a diverse toolkit comprising environment-sensitive small molecules, lipid-binding protein probes, hybrid chemical–genetic reporters and label-free imaging modalities has emerged to visualise membrane composition and its biophysical properties in living cells and tissues. Here, we highlight recent advances in (i) fluorescence-based readout of composition and membrane properties and (ii) mass spectrometry and vibrational imaging strategies for label-free spatial mapping of membrane composition and structure. We focus on sensing mechanisms, design principles and exemplar applications.