The intercalation of atoms, ions, and molecules is a powerful tool for altering or adjusting two-dimensional material properties (interlayer interactions, in-plane bonding configurations, Fermi level energy, electronic band structure, and spin orbit coupling). Insertion can cause changes in the properties of materials related to photonics, electronics, optoelectronics, thermoelectricity, magnetism, catalysis, and energy storage, releasing or enhancing the potential of two-dimensional materials in current and future applications. In situ imaging and spectroscopy techniques are used to visualize and track the intercalation process. These technologies provide opportunities to decipher important and often elusive intercalation dynamics, chemical mechanics, and mechanisms, such as intercalation pathways, reversibility, uniformity, and velocity. In this review, we discuss intercalation in two-dimensional materials, first briefly introducing intercalation strategies, then studying atomic and intrinsic effects of intercalation, followed by an overview of their in situ studies, and finally presenting our prospects.