Techniques such as flow cytometry and immunofluorescence histology have empowered many biological fields to draw strong conclusions about cellular events at a particular moment in time. However, definitive hypothesis testing can require in vivo confirmation of biological events. Multiphoton laser scanning microscopy (LSM) has proven to be an important modality for time-resolved imaging within live anesthetized animals. When appropriately configured, multiphoton LSM can enable the visualization of cellular behavior within the context of tissue architecture under physiologic conditions. This approach has been used by researchers to analyze the location and timing of cellular interactions, cell division, cell death or signaling as well as larger-scale shifts in tissue composition or structure. Many tissue types can be studied in this way including bone marrow, lymph nodes, intestines, lung, adipose tissue, and skin or tumors transplanted under the skin. The use of a pulsed multiphoton laser for fluorescence excitation is advantageous for in vivo imaging in part due to the ability of longer wavelengths to better penetrate tissue and its superb optical sectioning, allowing for excitation of fluorescence deeper in tissue with less phototoxicity over longer periods of time. The types of biological queries and analyses that would best benefit from this approach will be reviewed, along with its limitations and some of the requirements for its successful application.