Interfacial water associated with phospholipids plays an important role in different biological processes such as fibrillation of disordered protein, neurotransmitter interaction with lipids, and hydrated proton translocation. However, understanding the structural change of interfacial water and phospholipids during these biological processes is difficult due to the lack of appropriate selective technique and soft nature of these interfaces. Vibrational sum‐frequency generation (VSFG) is a second‐order nonlinear spectroscopic technique, which has the inherent surface selectivity as well as sensitivity and hence has been utilized to understand the properties of interfaces. In this review, the utilization of VSFG technique has been discussed in understanding the structural change of interfacial water and lipids during several biological processes.