Intravital imaging using two-photon laser scanning microscopy (TPSLM) can visualize the cellular dynamics in a living body. By recording spatiotemporal images, this technique allows the detection of unknown cell movements and related unknown phenomena in an environment in which blood circulation is maintained. Because of these advantages, this multidimensional in vivo analytical technique has attracted attention from various research fields. TPSLM has also been introduced in cancer research and utilized to visualize cancer cell movements. By determining cancer dynamics in vivo, it is expected that new cancer movement-related molecules will be discovered, which could be novel therapeutic targets. This article introduces the TPSLM imaging technique and method of molecular analysis using this imaging system.