3D cell culture systems such as organoids reflect the complexity of native organs more closely than traditional 2D culture systems. However, efforts to translate organoid research into clinical and industrial applications demand the development of compatible analytical platforms to accurately characterize their multifaceted physiological traits. This Review explores three vital aspects of physiological characterization - electrophysiological, biophysical and optical properties - including the role of omics and computational technologies in understanding organoid function and behaviour.