Since the rst demonstration of nonlinear imaging of biological samples in 1990 (Denk et al., 1990), multiphoton microscopy’s (MPM) applications in the eld of biomedical imaging is exponentially growing. is chapter focuses on giving an insight into the history and physical principles of nonlinear optics, distinct advantages MPM holds for biomedical application and its application in the organ-level characterization of liver brosis using liver biopsy and surface mapping. We envision that MPM will be a future tool for tissue engineering applications to monitor liver brosis progression and regression in demonstrating new therapies and regeneration of the liver.