Second Harmonic Generation (SHG) is a nonlinear optical technique that enables high-resolution, label-free imaging of non-centrosymmetric structures such as lipid membranes, collagen fibers, microtubules, and tissues. By applying a near-infrared laser, SHG generates signals at half the wavelength, facilitating specific interface imaging with minimal photodamage. This chapter begins with an explanation of the physical principles underlying SHG, highlighting why it is particularly effective for surface applications in biological samples. It then delves into the applications of SHG imaging, including pharmacokinetic studies of cell membranes, imaging of collagen fibers, and distinguishing tumor tissues from healthy tissues. The chapter concludes with a critical evaluation of SHG’s strengths—such as high specificity, non-invasive nature, and minimal sample preparation—and its limitations, including its restricted applicability to centrosymmetric molecules and potential signal interference. SHG imaging not only provides detailed insights into tissue structures independently but also, when combined with other imaging techniques, creates a comprehensive and detailed image of the tissue being studied. The advancement toward minimally invasive, tissue-preserving microscopy opens up expansive possibilities in diagnosing a wide range of diseases, potentially influencing therapeutic decisions. The capabilities of nonlinear optics in medicine have been demonstrated in recent years and are anticipated to be further harnessed as clinical application areas continue to evolve and expand.