Regulated exocytosis is a fundamental process that every secretory cell uses to deliver molecules to the cell surface and the extracellular space by virtue of membranous carriers. This process has been extensively studied using various approaches such as biochemistry, electrophysiology and electron microscopy. However, recent developments in time-lapse light microscopy have made possible imaging individual exocytic events, hence, advancing our understanding of this process at a molecular level. In this review, we focus on intravital microscopy (IVM), a light microscopy-based approach that enables imaging subcellular structures in live animals, and discuss its recent application to study regulated exocytosis. IVM has revealed differences in regulation and modality of regulated exocytosis between in vitro and in vivo model systems, unraveled novel aspects of this process that can be appreciated only in in vivo settings and provided valuable and novel information on its molecular machinery. In conclusion, we make the case for IVM being a mature technique that can be used to investigate the molecular machinery of several intracellular events under physiological conditions.