Most knowledge in the biological world has been gathered from studying images.1 Thus, a growing interest for live-tissue imaging has evolved. Fluorescence microscopy is highly sensitive and has good specificity, which makes it useful for studying the location and concentration of molecules and to view cellular events in real time.2,3 The first application of fluorescence microscopy in the liver occurred over 70 years ago. Since then it has provided vital information regarding normal and diseased liver function and morphology,4,5 vitamin A distribution,6 fat distribution,7 drug distribution,8,9 heterogeneity in drug distribution,10 and biliary function,11 to mention a few. In the last decade, these imaging techniques have been improved with confocal microscopy and nonlinear excitation microscopy, such as multiphoton microscopy (MPM). In conventional confocal microscopy, the intensity from the beam is approximately uniform above and below the focal plane, which results in the specimen generating fluorescence out of the focal plane that is rejected by the pinhole. This leads to the specimen being subjected to photobleaching and photodamage, affecting image quality and tissue health.2 MPM avoids this because a much smaller area of the specimen is being stimulated by the excitation light source and no out-of-focus light is generated, restricting photobleaching to the focal point only.2,12 Due to the excitation wavelength of MPM, 700 to 1000 nm in the near-infrared (NIR) range, at which the imaging penetration depth is maximized, tissue scattering and absorption are minimized.13,14 The most important application of MPM is to image the physiology, morphology, and cell–cell interactions of intact tissue of live animals with high resolution. Although one limitation is that it cannot quantitatively study cellular function on a molecular level,15 MPM in combination with fluorescence lifetime imaging microscopy (FLIM) can, however, identify fluorophores with overlapping spectral properties and measure lifetimes, which are specific for the fluorophore and the environment surrounding it.16 Fluorescence lifetime is the reciprocal sum of the rate constants of all possible return paths for the electron from the excited state to the ground state.17 One application for FLIM in liver imaging is to study the levels of autofluorescent nicotinamide adenine dinucleotide (NADH), as a direct measure of the metabolic state of the cells.18 This review aims to highlight the use of MPM in defining liver function, in particular, assessing morphology, metabolic activity, and mitochondrial function, as well as diagnosing cancer and fibrosis and drug transport in healthy and diseased livers.