Li-ion and Li-S batteries find enormous applications in different fields, such as electric vehicles and portable electronics. A separator is an indispensable part of the battery design, which functions as a physical barrier for the electrode as well as an electrolyte reservoir for ionic transport. The properties of the separators directly influence the performance of the batteries. Traditional polyolefin separators showed low thermal stability, poor wettability toward the electrolyte, and inadequate barrier properties to polysulfides. To improve the performance and durability of Li-ion and Li-S batteries, development of advanced separators is required. In this review, we summarize recent progress on the fabrication and application of novel separators, including the functionalized polyolefin separator, polymeric separator, and ceramic separator, for Li-ion and Li-S batteries. The characteristics, advantages, and limitations of these separators are discussed. A brief outlook for the future directions of the research in the separators is also provided.