Currently, two-photon uorescence (2PF) microscopy is the most common technique used in combination with second harmonic generation (SHG) microscopy. Making use of the various endogenous uorophores found in bio-tissues, 2PF microscopy can provide more cellular, morphological, and molecular information regarding these tissues in addition to the SHG-revealed information; moreover, it can also help localize the SHG signals and identify the contrast sources of these signals. However, since 2PF microscopy is a uorescence-based technique, issues of photodamage and photobleaching due to multiphoton absorption are always of concern in the imaging process. To avoid such uorescence-induced concerns, third harmonic generation (THG) microscopy, a third-order nonlinear optical microscopy, could be used instead of 2PF microscopy. THG microscopy is well known to have interface sensitivity and can be used as a general-purpose type of microscopy to provide structural information of the tissues. Based on the characteristics of virtual-level transition and energy conservation, which are the same as in SHG microscopy, with a combination of SHG with THG, the problems of uorescence-induced photodamage and photobleaching could be avoided so as to reduce the invasiveness. Meanwhile, owing to its higher-order nonlinearity, a higher spatial resolution can be achieved with THG microscopy. In this chapter, combined SHG/THG microscopy will be introduced, including the principles, system setup, and the biomedical applications accomplished.