Hair follicles progress through distinct phases of growth (anagen), regression (catagen), and rest (telogen), driven by complex cellular and molecular dynamics. Within the hair follicle niche, melanocyte stem cells (McSCs) serve as a critical reservoir for generating mature melanocytes responsible for hair pigmentation. A comprehensive understanding of McSC biology requires the ability to observe their behavior directly in live animals, a task complicated by the technical difficulties of long-term, high-resolution imaging. Here, we present a detailed protocol that utilizes intravital two-photon microscopy to overcome these challenges. We describe the complete workflow, from hair cycle synchronization via waxing to the setup and execution of longitudinal imaging sessions, allowing for the direct visualization of individual McSCs over weeks. This method is a powerful tool for investigating the fundamental dynamics of McSCs and serves as a platform to study how these processes are altered by external stressors or genetic modifications.